Yeming Wang

<h2>Summary</h2><h3>Background</h3> A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. <h3>Methods</h3> All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. <h3>Findings</h3> By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. <h3>Interpretation</h3> The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. <h3>Funding</h3> Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.

<h2>Summary</h2><h3>Background</h3> Since December, 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiological and clinical characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clinical course of illness, including viral shedding, have not been well described. <h3>Methods</h3> In this retrospective, multicentre cohort study, we included all adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demographic, clinical, treatment, and laboratory data, including serial samples for viral RNA detection, were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death. <h3>Findings</h3> 191 patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. 91 (48%) patients had a comorbidity, with hypertension being the most common (58 [30%] patients), followed by diabetes (36 [19%] patients) and coronary heart disease (15 [8%] patients). Multivariable regression showed increasing odds of in-hospital death associated with older age (odds ratio 1·10, 95% CI 1·03–1·17, per year increase; p=0·0043), higher Sequential Organ Failure Assessment (SOFA) score (5·65, 2·61–12·23; p<0·0001), and d-dimer greater than 1 μg/mL (18·42, 2·64–128·55; p=0·0033) on admission. Median duration of viral shedding was 20·0 days (IQR 17·0–24·0) in survivors, but SARS-CoV-2 was detectable until death in non-survivors. The longest observed duration of viral shedding in survivors was 37 days. <h3>Interpretation</h3> The potential risk factors of older age, high SOFA score, and d-dimer greater than 1 μg/mL could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future. <h3>Funding</h3> Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences; National Science Grant for Distinguished Young Scholars; National Key Research and Development Program of China; The Beijing Science and Technology Project; and Major Projects of National Science and Technology on New Drug Creation and Development.

No therapeutics have yet been proven effective for the treatment of severe illness caused by SARS-CoV-2.We conducted a randomized, controlled, open-label trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection, which causes the respiratory illness Covid-19, and an oxygen saturation (Sao2) of 94% or less while they were breathing ambient air or a ratio of the partial pressure of oxygen (Pao2) to the fraction of inspired oxygen (Fio2) of less than 300 mm Hg. Patients were randomly assigned in a 1:1 ratio to receive either lopinavir-ritonavir (400 mg and 100 mg, respectively) twice a day for 14 days, in addition to standard care, or standard care alone. The primary end point was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a seven-category ordinal scale or discharge from the hospital, whichever came first.A total of 199 patients with laboratory-confirmed SARS-CoV-2 infection underwent randomization; 99 were assigned to the lopinavir-ritonavir group, and 100 to the standard-care group. Treatment with lopinavir-ritonavir was not associated with a difference from standard care in the time to clinical improvement (hazard ratio for clinical improvement, 1.31; 95% confidence interval [CI], 0.95 to 1.80). Mortality at 28 days was similar in the lopinavir-ritonavir group and the standard-care group (19.2% vs. 25.0%; difference, -5.8 percentage points; 95% CI, -17.3 to 5.7). The percentages of patients with detectable viral RNA at various time points were similar. In a modified intention-to-treat analysis, lopinavir-ritonavir led to a median time to clinical improvement that was shorter by 1 day than that observed with standard care (hazard ratio, 1.39; 95% CI, 1.00 to 1.91). Gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. Lopinavir-ritonavir treatment was stopped early in 13 patients (13.8%) because of adverse events.In hospitalized adult patients with severe Covid-19, no benefit was observed with lopinavir-ritonavir treatment beyond standard care. Future trials in patients with severe illness may help to confirm or exclude the possibility of a treatment benefit. (Funded by Major Projects of National Science and Technology on New Drug Creation and Development and others; Chinese Clinical Trial Register number, ChiCTR2000029308.).

<h2>Summary</h2><h3>Background</h3> The long-term health consequences of COVID-19 remain largely unclear. The aim of this study was to describe the long-term health consequences of patients with COVID-19 who have been discharged from hospital and investigate the associated risk factors, in particular disease severity. <h3>Methods</h3> We did an ambidirectional cohort study of patients with confirmed COVID-19 who had been discharged from Jin Yin-tan Hospital (Wuhan, China) between Jan 7, 2020, and May 29, 2020. Patients who died before follow-up, patients for whom follow-up would be difficult because of psychotic disorders, dementia, or re-admission to hospital, those who were unable to move freely due to concomitant osteoarthropathy or immobile before or after discharge due to diseases such as stroke or pulmonary embolism, those who declined to participate, those who could not be contacted, and those living outside of Wuhan or in nursing or welfare homes were all excluded. All patients were interviewed with a series of questionnaires for evaluation of symptoms and health-related quality of life, underwent physical examinations and a 6-min walking test, and received blood tests. A stratified sampling procedure was used to sample patients according to their highest seven-category scale during their hospital stay as 3, 4, and 5–6, to receive pulmonary function test, high resolution CT of the chest, and ultrasonography. Enrolled patients who had participated in the Lopinavir Trial for Suppression of SARS-CoV-2 in China received severe acute respiratory syndrome coronavirus 2 antibody tests. Multivariable adjusted linear or logistic regression models were used to evaluate the association between disease severity and long-term health consequences. <h3>Findings</h3> In total, 1733 of 2469 discharged patients with COVID-19 were enrolled after 736 were excluded. Patients had a median age of 57·0 (IQR 47·0–65·0) years and 897 (52%) were men. The follow-up study was done from June 16, to Sept 3, 2020, and the median follow-up time after symptom onset was 186·0 (175·0–199·0) days. Fatigue or muscle weakness (63%, 1038 of 1655) and sleep difficulties (26%, 437 of 1655) were the most common symptoms. Anxiety or depression was reported among 23% (367 of 1617) of patients. The proportions of median 6-min walking distance less than the lower limit of the normal range were 24% for those at severity scale 3, 22% for severity scale 4, and 29% for severity scale 5–6. The corresponding proportions of patients with diffusion impairment were 22% for severity scale 3, 29% for scale 4, and 56% for scale 5–6, and median CT scores were 3·0 (IQR 2·0–5·0) for severity scale 3, 4·0 (3·0–5·0) for scale 4, and 5·0 (4·0–6·0) for scale 5–6. After multivariable adjustment, patients showed an odds ratio (OR) 1·61 (95% CI 0·80–3·25) for scale 4 versus scale 3 and 4·60 (1·85–11·48) for scale 5–6 versus scale 3 for diffusion impairment; OR 0·88 (0·66–1·17) for scale 4 versus scale 3 and OR 1·77 (1·05–2·97) for scale 5–6 versus scale 3 for anxiety or depression, and OR 0·74 (0·58–0·96) for scale 4 versus scale 3 and 2·69 (1·46–4·96) for scale 5–6 versus scale 3 for fatigue or muscle weakness. Of 94 patients with blood antibodies tested at follow-up, the seropositivity (96·2% <i>vs</i> 58·5%) and median titres (19·0 <i>vs</i> 10·0) of the neutralising antibodies were significantly lower compared with at the acute phase. 107 of 822 participants without acute kidney injury and with estimated glomerular filtration rate (eGFR) 90 mL/min per 1·73 m² or more at acute phase had eGFR less than 90 mL/min per 1·73 m² at follow-up. <h3>Interpretation</h3> At 6 months after acute infection, COVID-19 survivors were mainly troubled with fatigue or muscle weakness, sleep difficulties, and anxiety or depression. Patients who were more severely ill during their hospital stay had more severe impaired pulmonary diffusion capacities and abnormal chest imaging manifestations, and are the main target population for intervention of long-term recovery. <h3>Funding</h3> National Natural Science Foundation of China, Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, National Key Research and Development Program of China, Major Projects of National Science and Technology on New Drug Creation and Development of Pulmonary Tuberculosis, and Peking Union Medical College Foundation.

No specific antiviral drug has been proven effective for treatment of patients with severe coronavirus disease 2019 (COVID-19). Remdesivir (GS-5734), a nucleoside analogue prodrug, has inhibitory effects on pathogenic animal and human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, and inhibits Middle East respiratory syndrome coronavirus, SARS-CoV-1, and SARS-CoV-2 replication in animal models.We did a randomised, double-blind, placebo-controlled, multicentre trial at ten hospitals in Hubei, China. Eligible patients were adults (aged ≥18 years) admitted to hospital with laboratory-confirmed SARS-CoV-2 infection, with an interval from symptom onset to enrolment of 12 days or less, oxygen saturation of 94% or less on room air or a ratio of arterial oxygen partial pressure to fractional inspired oxygen of 300 mm Hg or less, and radiologically confirmed pneumonia. Patients were randomly assigned in a 2:1 ratio to intravenous remdesivir (200 mg on day 1 followed by 100 mg on days 2-10 in single daily infusions) or the same volume of placebo infusions for 10 days. Patients were permitted concomitant use of lopinavir-ritonavir, interferons, and corticosteroids. The primary endpoint was time to clinical improvement up to day 28, defined as the time (in days) from randomisation to the point of a decline of two levels on a six-point ordinal scale of clinical status (from 1=discharged to 6=death) or discharged alive from hospital, whichever came first. Primary analysis was done in the intention-to-treat (ITT) population and safety analysis was done in all patients who started their assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04257656.Between Feb 6, 2020, and March 12, 2020, 237 patients were enrolled and randomly assigned to a treatment group (158 to remdesivir and 79 to placebo); one patient in the placebo group who withdrew after randomisation was not included in the ITT population. Remdesivir use was not associated with a difference in time to clinical improvement (hazard ratio 1·23 [95% CI 0·87-1·75]). Although not statistically significant, patients receiving remdesivir had a numerically faster time to clinical improvement than those receiving placebo among patients with symptom duration of 10 days or less (hazard ratio 1·52 [0·95-2·43]). Adverse events were reported in 102 (66%) of 155 remdesivir recipients versus 50 (64%) of 78 placebo recipients. Remdesivir was stopped early because of adverse events in 18 (12%) patients versus four (5%) patients who stopped placebo early.In this study of adult patients admitted to hospital for severe COVID-19, remdesivir was not associated with statistically significant clinical benefits. However, the numerical reduction in time to clinical improvement in those treated earlier requires confirmation in larger studies.Chinese Academy of Medical Sciences Emergency Project of COVID-19, National Key Research and Development Program of China, the Beijing Science and Technology Project.

The full range of long-term health consequences of COVID-19 in patients who are discharged from hospital is largely unclear. The aim of our study was to comprehensively compare consequences between 6 months and 12 months after symptom onset among hospital survivors with COVID-19.We undertook an ambidirectional cohort study of COVID-19 survivors who had been discharged from Jin Yin-tan Hospital (Wuhan, China) between Jan 7 and May 29, 2020. At 6-month and 12-month follow-up visit, survivors were interviewed with questionnaires on symptoms and health-related quality of life (HRQoL), and received a physical examination, a 6-min walking test, and laboratory tests. They were required to report their health-care use after discharge and work status at the 12-month visit. Survivors who had completed pulmonary function tests or had lung radiographic abnormality at 6 months were given the corresponding tests at 12 months. Non-COVID-19 participants (controls) matched for age, sex, and comorbidities were interviewed and completed questionnaires to assess prevalent symptoms and HRQoL. The primary outcomes were symptoms, modified British Medical Research Council (mMRC) score, HRQoL, and distance walked in 6 min (6MWD). Multivariable adjusted logistic regression models were used to evaluate the risk factors of 12-month outcomes.1276 COVID-19 survivors completed both visits. The median age of patients was 59·0 years (IQR 49·0-67·0) and 681 (53%) were men. The median follow-up time was 185·0 days (IQR 175·0-198·0) for the 6-month visit and 349·0 days (337·0-361·0) for the 12-month visit after symptom onset. The proportion of patients with at least one sequelae symptom decreased from 68% (831/1227) at 6 months to 49% (620/1272) at 12 months (p<0·0001). The proportion of patients with dyspnoea, characterised by mMRC score of 1 or more, slightly increased from 26% (313/1185) at 6-month visit to 30% (380/1271) at 12-month visit (p=0·014). Additionally, more patients had anxiety or depression at 12-month visit (26% [331/1271] at 12-month visit vs 23% [274/1187] at 6-month visit; p=0·015). No significant difference on 6MWD was observed between 6 months and 12 months. 88% (422/479) of patients who were employed before COVID-19 had returned to their original work at 12 months. Compared with men, women had an odds ratio of 1·43 (95% CI 1·04-1·96) for fatigue or muscle weakness, 2·00 (1·48-2·69) for anxiety or depression, and 2·97 (1·50-5·88) for diffusion impairment. Matched COVID-19 survivors at 12 months had more problems with mobility, pain or discomfort, and anxiety or depression, and had more prevalent symptoms than did controls.Most COVID-19 survivors had a good physical and functional recovery during 1-year follow-up, and had returned to their original work and life. The health status in our cohort of COVID-19 survivors at 12 months was still lower than that in the control population.Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, the National Natural Science Foundation of China, the National Key Research and Development Program of China, Major Projects of National Science and Technology on New Drug Creation and Development of Pulmonary Tuberculosis, the China Evergrande Group, Jack Ma Foundation, Sino Biopharmaceutical, Ping An Insurance (Group), and New Sunshine Charity Foundation.

Rapidly progressing hypoxemia and acute respiratory distress syndrome were commonly observed in patients with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) viral pneumonia [1]. Although several severity scores including Pneumonia Severity Index (PSI) [2], CURB-65 and CRB-65 (confusion, (urea >7 mmol·L−1), respiratory rate ≥30 breaths·min−1, blood pressure <90 mmHg (systolic) ≤60 mmHg (diastolic), age ≥65 years), [3], A-DROP [4] and SMART-COP [5] have been developed to identify community acquired pneumonia (CAP) patients at high risk and offer therapeutic advice, the underestimation of risk of death from viral pneumonia in these scores has been reported by previous studies [6, 7]. The National Early Warning Score 2 (NEWS2) was developed by National Health Service (NHS) England [8] and, along with quick sequential organ failure assessment score (qSOFA), was proposed as a candidate for prognostic prediction for severe coronavirus disease 2019 (COVID-19) in the situation of limited medical source [9]. The aim of this study was to compare the accuracy of current score rules in hospitalised patients with COVID-19 pneumonia for predicting the risk of death and evaluate feasibility in improving medical decisions by adopting appropriate scores in clinical practice. A-DROP is a reliable tool for risk stratification of death in COVID-19 hospitalised patients on admission <https://bit.ly/3iDZipD> We acknowledge all healthcare workers involved in the diagnosis and treatment of patients in Wuhan, China.

The memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection.In this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms.We enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection.SARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time.Chinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council.

Abstract Background A synergistic effect of combination therapy with favipiravir and oseltamivir has been reported in preclinical models of influenza. However, no data are available on the clinical effectiveness of combination therapy in severe influenza. Methods Data from 2 separate prospective studies of influenza adults were used to compare outcomes between combination and oseltamivir monotherapy. Outcomes included rate of clinical improvement (defined as a decrease of 2 categories on a 7-category ordinal scale) and viral RNA detectability over time. Subhazard ratios (sHRs) were estimated by the Fine and Gray model for competing risks. Results In total, 40 patients were treated with combination therapy and 128 with oseltamivir alone. Clinical improvement on day 14 in the combination group was higher than in the monotherapy group (62.5% vs 42.2%; P = .0247). The adjusted sHR for combination therapy was 2.06 (95% confidence interval, 1.30–3.26). The proportion of undetectable viral RNA at day 10 was higher in the combination group than the oseltamivir group (67.5% vs 21.9%; P &amp;lt; .01). No significant differences were observed in mortality or other outcomes. Conclusions Favipiravir and oseltamivir combination therapy may accelerate clinical recovery compared to oseltamivir monotherapy in severe influenza, and this strategy should be formally evaluated in a randomized controlled trial.

The recent outbreak of respiratory illness in Wuhan, China is caused by a novel coronavirus, named 2019-nCoV, which is genetically close to a bat-derived coronavirus. 2019-nCoV is categorized as beta genus coronavirus, same as the two other strains - severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Antiviral drugs commonly used in clinical practice, including neuraminidase inhibitors (oseltamivir, paramivir, zanamivir, etc.), ganciclovir, acyclovir and ribavirin, are invalid for 2019-nCoV and not recommended. Drugs are possibly effective for 2019-nCoV include: remdesivir, lopinavir / ritonavir, lopinavir / ritonavir combined with interferon-β, convalescent plasma, and monoclonal antibodies. But the efficacy and safety of these drugs for 2019-nCoV pneumonia patients need to be assessed by further clinical trials.2019新型冠状病毒(2019-nCoV)是武汉不明原因肺炎的致病原。2019-nCoV在遗传学上与一种蝙蝠来源的新型冠状病毒比较接近，与SARS-CoV、MERS-CoV同为β属冠状病毒。目前临床上常用的抗病毒药物，包括神经氨酸酶抑制剂（奥司他韦、帕拉米韦、扎那米韦等）、更昔洛韦、阿昔洛韦、利巴韦林等药物对2019-nCoV均无效，不建议临床应用。目前研究证实可能有效的药物包括：瑞德西韦、洛匹那韦/利托那韦、洛匹那韦/利托那韦联合干扰素-β、恢复期血浆、单克隆抗体。但这些药物在2019-nCoV肺炎患者中的疗效和安全性有待进一步临床实验证实。.

Seasonal human coronaviruses (HCoVs) including HCoV-229E, -OC43, -NL63, and -HKU1 widely spread in global human populations. However, the relevance of humoral response against seasonal HCoVs to COVID-19 pathogenesis is elusive. In this study, we profiled the temporal changes of IgG antibody against spike proteins (S-IgG) of SARS-CoV-2 and seasonal HCoVs in 838 plasma samples collected from 344 COVID-19 patients. We tested the antigenic cross-reactivities of S protein between SARS-CoV-2 and seasonal HCoVs and evaluated the correlations between the levels of HCoV-OC43 S-IgG and the disease severity in COVID-19 patients. We found that SARS-CoV-2 S-IgG titres mounted until days 22–28, whereas HCoV-OC43 antibody titres increased until days 15–21 and then plateaued until day 46. However, IgG titres against HCoV-NL63, −229E, and -HKU1 showed no significant increase. A two-way cross-reactivity was identified between SARS-CoV-2 and HCoV-OC43. Neutralizing antibodies against SARS-CoV-2 were not detectable in healthy controls who were positive for HCoV-OC43 S-IgG. HCoV-OC43 S-IgG titres were significantly higher in patients with severe disease than those in mild patients at days 1–21 post symptom onset (PSO). Higher levels of HCoV-OC43 S-IgG were also observed in patients requiring mechanical ventilation. At days 1–10 PSO, HCoV-OC43 S-IgG titres correlated to disease severity in the age group over 60. Our data indicate that there is a correlation between cross-reactive antibody against HCoV-OC43 spike protein and disease severity in COVID-19 patients.

The long-term health consequences of COVID-19 remain largely unclear. The aim of this study was to describe the long-term health consequences of patients with COVID-19 who have been discharged from hospital and investigate the associated risk factors, in particular disease severity.We did an ambidirectional cohort study of patients with confirmed COVID-19 who had been discharged from Jin Yin-tan Hospital (Wuhan, China) between Jan 7 and May 29, 2020. Patients who died before follow-up; patients for whom follow-up would be difficult because of psychotic disorders, dementia, or readmission to hospital; those who were unable to move freely due to concomitant osteoarthropathy or immobile before or after discharge due to diseases such as stroke or pulmonary embolism; those who declined to participate; those who could not be contacted; and those living outside of Wuhan or in nursing or welfare homes were all excluded. All patients were interviewed with a series of questionnaires for evaluation of symptoms and health-related quality of life, underwent physical examinations and a 6-min walking test, and received blood tests. A stratified sampling procedure was used to sample patients according to their highest seven-category scale during their hospital stay as 3, 4, and 5-6, to receive pulmonary function test, high resolution CT of the chest, and ultrasonography. Enrolled patients who had participated in the Lopinavir Trial for Suppression of SARS-CoV-2 in China received SARS-CoV-2 antibody tests. Multivariable adjusted linear or logistic regression models were used to evaluate the association between disease severity and long-term health consequences.In total, 1733 of 2469 discharged patients with COVID-19 were enrolled after 736 were excluded. Patients had a median age of 57·0 years (IQR 47·0-65·0) and 897 (52%) were male and 836 (48%) were female. The follow-up study was done from June 16 to Sept 3, 2020, and the median follow-up time after symptom onset was 186·0 days (175·0-199·0). Fatigue or muscle weakness (52%, 855 of 1654) and sleep difficulties (26%, 437 of 1655) were the most common symptoms. Anxiety or depression was reported among 23% (367 of 1616) of patients. The proportions of 6-min walking distance less than the lower limit of the normal range were 17% for those at severity scale 3, 13% for severity scale 4, and 28% for severity scale 5-6. The corresponding proportions of patients with diffusion impairment were 22% for severity scale 3, 29% for scale 4, and 56% for scale 5-6, and median CT scores were 3·0 (IQR 2·0-5·0) for severity scale 3, 4·0 (3·0-5·0) for scale 4, and 5·0 (4·0-6·0) for scale 5-6. After multivariable adjustment, patients showed an odds ratio (OR) of 1·61 (95% CI 0·80-3·25) for scale 4 versus scale 3 and 4·60 (1·85-11·48) for scale 5-6 versus scale 3 for diffusion impairment; OR 0·88 (0·66-1·17) for scale 4 versus scale 3 and OR 1·76 (1·05-2·96) for scale 5-6 versus scale 3 for anxiety or depression, and OR 0·87 (0·68-1·11) for scale 4 versus scale 3 and 2·75 (1·61-4·69) for scale 5-6 versus scale 3 for fatigue or muscle weakness. Of 94 patients with blood antibodies tested at follow-up, the seropositivity (96·2% vs 58·5%) and median titres (19·0 vs 10·0) of the neutralising antibodies were significantly lower compared with at the acute phase. 107 of 822 participants without acute kidney injury and with an estimated glomerular filtration rate (eGFR) of 90 mL/min per 1·73 m2 or more at acute phase had eGFR less than 90 mL/min per 1·73 m2 at follow-up.At 6 months after acute infection, COVID-19 survivors were mainly troubled with fatigue or muscle weakness, sleep difficulties, and anxiety or depression. Patients who were more severely ill during their hospital stay had more severe impaired pulmonary diffusion capacities and abnormal chest imaging manifestations, and are the main target population for intervention of long-term recovery.National Natural Science Foundation of China, Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, National Key Research and Development Program of China, Major Projects of National Science and Technology on New Drug Creation and Development of Pulmonary Tuberculosis, and Peking Union Medical College Foundation.

BackgroundInterpretation of the evidence from randomised controlled trials (RCTs) of remdesivir in patients treated in hospital for COVID-19 is conflicting. We aimed to assess the benefits and harms of remdesivir compared with placebo or usual care in these patients, and whether treatment effects differed between prespecified patient subgroups.MethodsFor this systematic review and meta-analysis, we searched PubMed, Embase, the Cochrane COVID-19 trial registry, ClinicalTrials.gov, the International Clinical Trials Registry Platform, and preprint servers from Jan 1, 2020, until April 11, 2022, for RCTs of remdesivir in adult patients hospitalised with COVID-19, and contacted the authors of eligible trials to request individual patient data. The primary outcome was all-cause mortality at day 28 after randomisation. We used multivariable hierarchical regression—adjusting for respiratory support, age, and enrollment period—to investigate effect modifiers. This study was registered with PROSPERO, CRD42021257134.FindingsOur search identified 857 records, yielding nine RCTs eligible for inclusion. Of these nine eligible RCTs, individual data were provided for eight, covering 10 480 patients hospitalised with COVID-19 (99% of such patients included in such RCTs worldwide) recruited between Feb 6, 2020, and April 1, 2021. Within 28 days of randomisation, 662 (12·5%) of 5317 patients assigned to remdesivir and 706 (14·1%) of 5005 patients assigned to no remdesivir died (adjusted odds ratio [aOR] 0·88, 95% CI 0·78–1·00, p=0·045). We found evidence for a credible subgroup effect according to respiratory support at baseline (pinteraction=0·019). Of patients who were ventilated—including those who received high-flow oxygen—253 (30·0%) of 844 patients assigned to remdesivir died compared with 241 (28·5%) of 846 patients assigned to no remdesivir (aOR 1·10 [0·88–1·38]; low-certainty evidence). Of patients who received no oxygen or low-flow oxygen, 409 (9·1%) of 4473 patients assigned to remdesivir died compared with 465 (11·2%) of 4159 patients assigned to no remdesivir (0·80 [0·70–0·93]; high-certainty evidence). No credible subgroup effect was found for time to start of remdesivir after symptom onset, age, presence of comorbidities, enrolment period, or corticosteroid use. Remdesivir did not increase the frequency of severe or serious adverse events.InterpretationThis individual patient data meta-analysis showed that remdesivir reduced mortality in patients hospitalised with COVID-19 who required no or conventional oxygen support, but was underpowered to evaluate patients who were ventilated when receiving remdesivir. The effect size of remdesivir in patients with more respiratory support or acquired immunity and the cost-effectiveness of remdesivir remain to be further elucidated.FundingThe European Union's Horizon 2020 research and innovation programme.

Simnotrelvir is an oral 3-chymotrypsin-like protease inhibitor that has been found to have in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential efficacy in a phase 1B trial.In this phase 2-3, double-blind, randomized, placebo-controlled trial, we assigned patients who had mild-to-moderate coronavirus disease 2019 (Covid-19) and onset of symptoms within the past 3 days in a 1:1 ratio to receive 750 mg of simnotrelvir plus 100 mg of ritonavir or placebo twice daily for 5 days. The primary efficacy end point was the time to sustained resolution of symptoms, defined as the absence of 11 Covid-19-related symptoms for 2 consecutive days. Safety and changes in viral load were also assessed.A total of 1208 patients were enrolled at 35 sites in China; 603 were assigned to receive simnotrelvir and 605 to receive placebo. Among patients in the modified intention-to-treat population who received the first dose of trial drug or placebo within 72 hours after symptom onset, the time to sustained resolution of Covid-19 symptoms was significantly shorter in the simnotrelvir group than in the placebo group (180.1 hours [95% confidence interval {CI}, 162.1 to 201.6] vs. 216.0 hours [95% CI, 203.4 to 228.1]; median difference, -35.8 hours [95% CI, -60.1 to -12.4]; P = 0.006 by Peto-Prentice test). On day 5, the decrease in viral load from baseline was greater in the simnotrelvir group than in the placebo group (mean difference [±SE], -1.51±0.14 log10 copies per milliliter; 95% CI, -1.79 to -1.24). The incidence of adverse events during treatment was higher in the simnotrelvir group than in the placebo group (29.0% vs. 21.6%). Most adverse events were mild or moderate.Early administration of simnotrelvir plus ritonavir shortened the time to the resolution of symptoms among adult patients with Covid-19, without evident safety concerns. (Funded by Jiangsu Simcere Pharmaceutical; ClinicalTrials.gov number, NCT05506176.).

Background There is a paucity of data on the natural trajectory of outcomes in survivors of COVID-19 beyond 2 years after symptom onset, and no evidence exists on the effect of re-infection in people with long COVID symptoms. We aimed to investigate the 3-year health outcomes of COVID-19 survivors and the effect of omicron re-infection. Methods In this single-centre, longitudinal cohort study, we recruited participants with confirmed COVID-19 who were discharged from the Jin Yin-tan hospital in Wuhan, China, between Jan 7 and May 29, 2020. Participants completed three follow-up visits at 6 months (June 16 to Sept 13, 2020), 1 year (Dec 16, 2020, to Feb 7, 2021), and 2 years (Nov 16, 2021, to Jan 10, 2022) since symptom onset (reported previously). At 1-year follow-up, community controls without a history of SARS-CoV-2 infection were recruited from two communities in Wuhan and at 2 years were matched (1:1) with survivors of COVID-19 who underwent pulmonary function tests. We did a 3-year follow-up from Feb 23, 2023, to April 20, 2023, after the omicron (B.1.1.529) wave in winter, 2022. All eligible survivors of COVID-19 and community controls matched at 2-year follow-up were invited to the outpatient clinic at the hospital to complete several face-to-face questionnaires, a 6-min walking test (6MWT), and laboratory tests. A subgroup of survivors of COVID-19 identified by stratified sampling on the basis of disease severity scale score during hospitalisation and community controls underwent pulmonary function tests. Survivors of COVID-19 who received high-resolution CT and showed abnormal lung images at 2-year follow-up were invited for another assessment. We identified participants with and without long COVID at 2 years. The primary outcomes were sequelae symptoms, omicron infection, lung function, and chest imaging at the 3-year follow-up. Findings Of 1359 COVID-19 survivors who completed 2-year and 3-year follow-up, 728 (54%) had at least one sequelae symptom at 3 years after symptom onset and before omicron infection, mainly mild to moderate severity. During the omicron wave, participants with long COVID at 2 years had a significantly higher proportion of re-infection (573 [76%] of 753 vs 409 [67%] of 606 without long COVID; p=0·0004), pneumonia (27 [5%] of 568 vs seven [2%] of 403; p=0·012). 3 months after omicron infection, 126 (62%) of 204 survivors with long COVID at 2 years had newly occurring or worse symptoms, which was significantly higher than the proportion in the non-long COVID group (85 [41%] of 205; p<0·0001) and community controls (81 [40%] of 205; p<0·0001), and not significantly different between COVID-19 survivors without long COVID and matched community controls (85 [41%] of 205 vs 81 [39%] of 206; p=0·66). Re-infection was a risk factor for dyspnoea (odds ratio 1·36 [95% CI 1·04 to 1·77]; p=0·023), anxiety or depression (OR 1·65 [1·24 to 2·20]; p=0·0007), EuroQol visual analogue scale score (β –4·51 [–6·08 to –2·95]; p<0·0001), but not for reduced daily activity (0·72 [0·38 to 1·37]; p=0·32) at 3 years. Lung function of survivors at 3 years was similar to that of matched community controls. We found irregular line, traction bronchiectasis, subpleural lines and ground glass opacity at 3 years, but the volume ratio of lung lesion to total lung was only 0·2–0·3%. Interpretation Most long COVID symptoms at 3 years were mild to moderate, with lung function recovering to levels of matched controls. Survivors with long COVID had a higher proportion of participants with re-infection and newly occurring or worse symptoms 3 months after omicron infection than those without long COVID. Re-infection had increased symptom occurrence but not increased reduced daily activity. Although the organ function of survivors of COVID-19 recovered over time, those with severe long COVID symptoms, abnormal organ function, or limited mobility require urgent attention in future clinical practice and research. Funding Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, National Natural Science Foundation of China.

Guanosine tetraphosphate (ppGpp) is an alarmone that enables bacteria to adapt to their environment. It has been known for years that ppGpp acts directly on RNA polymerase (RNAP) to alter the rate of transcription, but its exact target site is still under debate. Here we report a crystal structure of Escherichia coli RNAP holoenzyme in complex with ppGpp at 4.5 Å resolution. The structure reveals that ppGpp binds at an interface between the shelf and core modules on the outer surface of RNAP, away from the catalytic center and the nucleic acid binding path. Bound ppGpp connects these two pivotal modules that may restrain the opening of the RNAP cleft. A detailed mechanism of action of ppGpp is proposed in which ppGpp prevents the closure of the active center that is induced by the binding of NTP, which could slow down nucleotide addition cycles and destabilize the initial transcription complexes.

Objective: To evaluate the clinical, radiographic, and functional outcomes of middle and distal third humeral shaft fractures treated with the minimally invasive percutaneous osteosynthesis (MIPO). Design: Prospective, single-center, nonconsecutive clinical series study. Setting: Skeletal trauma center of a university teaching hospital. Patients: Thirteen patients who were seen from May 2004 to October 2005 with an average age of 38.1 years (range, 25 to 60 years) form the basis of this study. Patients were obtained from a surgical database of 1 surgeon. Intervention: The middle and the distal third humeral shaft fractures were reduced by closed means and fixed with long narrow 4.5-mm dynamic compression plates introduced through 2 small incisions away from the fracture sites and placed on the anterior aspect of the humerus. Main Outcome Measurements: Time to fracture healing and functional assessments were assessed at an average follow-up of 12.5 months (range, 7 to 19 months) for the affected shoulders and elbows using the UCLA and Mayo elbow performance scoring systems, respectively. Results: All fractures united with a mean healing time of 16.2 weeks (range, 12 to 32 years). There were no nonunions, radial nerve palsies, or implant failures. The UCLA scoring system showed excellent results in 7 cases (53.8%) and good results in 6 cases (46.2%). Thirteen patients had excellent results of their elbow function when assessed with the Mayo elbow performance scoring system. Conclusion: Closed reduction and internal fixation of middle or distal third humeral shaft fractures using MIPO is a safe and effective surgical treatment method and an alternative option to open techniques.

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs (miRNAs) from pre-miRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage and an N-terminal helicase motif, whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.

Objectives: To evaluate the prevalence of cardiac injury and its association with mortality in hospitalized patients infected with avian influenza A (H7N9) virus. Design: Retrospective cohort study. Setting: A total of 133 hospitals in 17 provinces, autonomous regions, and municipalities of mainland China that admitted influenza A (H7N9) virus–infected patients between January 22, 2015, and June 16, 2017. Patients: A total of 321 patients with influenza A (H7N9) virus infection were included in the final analysis. Interventions: None. Measurements and Main Results: Demographics and clinical characteristics were collected from medical records. Cardiac injury was defined according to cardiac biomarkers, electrocardiography, or echocardiography. Among the 321 patients, 203 (63.2%) showed evidence of cardiac injury. Compared with the uninjured group, the cardiac injury group had lower Pa o 2 /F io 2 (median, 102.0 vs 148.4 mm Hg; p &lt; 0.001), higher Acute Physiology and Chronic Health Evaluation II score (median, 17.0 vs 11.0; p &lt; 0.001), longer stay in the ICU (10.0 vs 9.0 d; p = 0.029), and higher proportion of in-hospital death (64.0% vs 20.3%; p &lt; 0.001). The proportion of virus clearance until discharge or death was lower in the cardiac injury group than in the uninjured group (58.6% vs 86.4%; p &lt; 0.001). Multivariable-adjusted Cox proportional hazards regression analysis showed that cardiac injury was associated with higher mortality (hazards ratio, 2.06; 95% CI, 1.31–3.24) during hospitalization. Conclusions: Cardiac injury is a frequent condition among hospitalized patients infected with influenza A (H7N9) virus, and it is associated with higher risk of mortality.

Data are limited on the impact of neuraminidase inhibitor (NAI) treatment on avian influenza A(H7N9) virus RNA shedding. In this multicenter, retrospective study, data were collected from adults hospitalized with A(H7N9) infection during 2013–2017 in China. We compared clinical features and A(H7N9) shedding among patients with different NAI doses and combination therapies and evaluated factors associated with A(H7N9) shedding, using Cox proportional hazards regression. Among 478 patients, the median age was 56 years, 71% were male, and 37% died. The median time from illness onset to NAI treatment initiation was 8 days (interquartile range [IQR], 6–10 days), and the median duration of A(H7N9) RNA detection from onset was 15.5 days (IQR, 12–20 days). A(H7N9) RNA shedding was shorter in survivors than in patients who died (P < .001). Corticosteroid administration (hazard ratio [HR], 0.62 [95% confidence interval {CI}, .50–.77]) and delayed NAI treatment (HR, 0.90 [95% CI, .91–.96]) were independent risk factors for prolonged A(H7N9) shedding. There was no significant difference in A(H7N9) shedding duration between NAI combination treatment and monotherapy (P = .65) or between standard-dose and double-dose oseltamivir treatment (P = .70). Corticosteroid therapy and delayed NAI treatment were associated with prolonged A(H7N9) RNA shedding. NAI combination therapy and double-dose oseltamivir treatment were not associated with a reduced A(H7N9) shedding duration as compared to standard-dose oseltamivir.

Endpoint choice for randomized controlled trials of treatments for novel coronavirus-induced disease (COVID-19) is complex. Trials must start rapidly to identify treatments that can be used as part of the outbreak response, in the midst of considerable uncertainty and limited information. COVID-19 presentation is heterogeneous, ranging from mild disease that improves within days to critical disease that can last weeks to over a month and can end in death. While improvement in mortality would provide unquestionable evidence about the clinical significance of a treatment, sample sizes for a study evaluating mortality are large and may be impractical, particularly given a multitude of putative therapies to evaluate. Furthermore, patient states in between "cure" and "death" represent meaningful distinctions. Clinical severity scores have been proposed as an alternative. However, the appropriate summary measure for severity scores has been the subject of debate, particularly given the variable time course of COVID-19. Outcomes measured at fixed time points, such as a comparison of severity scores between treatment and control at day 14, may risk missing the time of clinical benefit. An endpoint such as time to improvement (or recovery) avoids the timing problem. However, some have argued that power losses will result from reducing the ordinal scale to a binary state of "recovered" versus "not recovered."We evaluate statistical power for possible trial endpoints for COVID-19 treatment trials using simulation models and data from two recent COVID-19 treatment trials.Power for fixed time-point methods depends heavily on the time selected for evaluation. Time-to-event approaches have reasonable statistical power, even when compared with a fixed time-point method evaluated at the optimal time.Time-to-event analysis methods have advantages in the COVID-19 setting, unless the optimal time for evaluating treatment effect is known in advance. Even when the optimal time is known, a time-to-event approach may increase power for interim analyses.

Coronavirus disease 2019 (COVID-19), caused by a novel corinavirus (later named SARS-CoV-2 virus), was fistly reported in Wuhan, Hubei Province, China towards the end of 2019. Large-scale spread within China and internationally led the World Health Organization to declare a Public Health Emergency of International Concern on 30th January 2020. The clinical manifestations of COVID-19 virus infection include asymptomatic infection, mild upper respiratory symptoms, severe viral pneumonia with respiratory failure, and even death. There are no antivirals of proven clinical efficacy in coronavirus infections. Remdesivir (GS-5734), a nucleoside analogue, has inhibitory effects on animal and human highly pathogenic coronaviruses, including MERS-CoV and SARS-CoV, in in vitro and in vivo experiments. It is also inhibitory against the COVID-19 virus in vitro. The aim of this study is to assess the efficacy and safety of remdesivir in adult patients with severe COVID-19.The protocol is prepared in accordance with the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) guidelines. This is a phase 3, randomized, double-blind, placebo-controlled, multicentre trial. Adults (≥ 18 years) with laboratory-confirmed COVID-19 virus infection, severe pneumonia signs or symptoms, and radiologically confirmed severe pneumonia are randomly assigned in a 2:1 ratio to intravenously administered remdesivir or placebo for 10 days. The primary endpoint is time to clinical improvement (censored at day 28), defined as the time (in days) from randomization of study treatment (remdesivir or placebo) until a decline of two categories on a six-category ordinal scale of clinical status (1 = discharged; 6 = death) or live discharge from hospital. One interim analysis for efficacy and futility will be conducted once half of the total number of events required has been observed.This is the first randomized, placebo-controlled trial in COVID-19. Enrolment began in sites in Wuhan, Hubei Province, China on 6th February 2020.ClinicalTrials.gov: NCT04257656. Registered on 6 February 2020.

Rationale: Alteration of human respiratory microbiota had been observed in coronavirus disease (COVID-19). How the microbiota is associated with the prognosis in COVID-19 is unclear. Objectives: To characterize the feature and dynamics of the respiratory microbiota and its associations with clinical features in patients with COVID-19. Methods: We conducted metatranscriptome sequencing on 588 longitudinal oropharyngeal swab specimens collected from 192 patients with COVID-19 (including 39 deceased patients) and 95 healthy controls from the same geographic area. Meanwhile, the concentration of 27 cytokines and chemokines in plasma was measured for patients with COVID-19. Measurements and Main Results: The upper respiratory tract (URT) microbiota in patients with COVID-19 differed from that in healthy controls, whereas deceased patients possessed a more distinct microbiota, both on admission and before discharge/death. The alteration of URT microbiota showed a significant correlation with the concentration of proinflammatory cytokines and mortality. Specifically, Streptococcus-dominated microbiota was enriched in recovered patients, and showed high temporal stability and resistance against pathogens. In contrast, the microbiota in deceased patients was more susceptible to secondary infections and became more deviated from the norm after admission. Moreover, the abundance of S. parasanguinis on admission was significantly correlated with prognosis in nonsevere patients (lower vs. higher abundance, odds ratio, 7.80; 95% CI, 1.70–42.05). Conclusions: URT microbiota dysbiosis is a remarkable manifestation of COVID-19; its association with mortality suggests it may reflect the interplay between pathogens, symbionts, and the host immune status. Whether URT microbiota could be used as a biomarker for diagnosis and prognosis of respiratory diseases merits further investigation.

Striking similarities have been found between coronavirus disease 2019 (COVID-19) and anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab)-related dermatomyositis, implying a shared autoinflammatory aberrance. Herein, we aim to investigate whether the anti-MDA5 Ab is present in COVID-19 and correlates with the severity and adverse outcome of COVID-19 patients.We retrospectively recruited 274 adult inpatients with COVID-19 in this study, including 48, 164, and 62 cases of deaths, severe, and non-severe patients respectively. The anti-MDA5 Ab was determined by ELISA and verified by Western Blotting, which indicated that the positive rate of anti-MDA5 Ab in COVID-19 patients was 48.2% (132/274). The clinical and laboratory features, as well as outcomes between patients with positive and negative anti-MDA5 Ab were compared and we found that the anti-MDA5 Ab positive patients tended to represent severe disease (88.6% vs 66.9%, P<0.0001). We also demonstrated that the titer of anti-MDA5 Ab was significantly elevated in the non-survivals (5.95 ± 5.16 vs 8.22 ± 6.64, P=0.030) and the positive rate was also higher than that in the survivals (23.5% vs 12.0%, P=0.012). Regarding severe COVID-19 patients, we found that high titer of anti-MDA5 Ab (≥10.0 U/mL) was more prevalent in the non-survivals (31.2% vs 14.0%, P=0.006). Moreover, a dynamic analysis of anti-MDA5 Ab was conducted at different time-points of COVID-19, which revealed that early profiling of anti-MDA5 Ab could distinguish severe patients from those with non-severe ones.Anti-MDA5 Ab was prevalent in the COVID-19 patients and high titer of this antibody is correlated with severe disease and unfavorable outcomes.

Kidney damage in COVID-19 patients has been of special concern. The association of acute kidney injury (AKI) with post-acute kidney function among COVID-19 survivors was not sufficiently elucidated.An ambidirectional cohort study was conducted with enrollment of COVID-19 survivors discharged from hospital between Jan 7, and May 29, 2020. Study participants were invited to follow-up visits at 6 and 12 months after symptom onset. The primary outcome was percentage of estimated glomerular filtration rate (eGFR) decreased from acute phase (between symptom onset and hospital discharge) to follow-up, and secondary outcome was reduced renal function at follow-up.In total, 1,734 study participants were included in this study. Median follow-up duration was 342.0 days (IQR, 223.0-358.0) after symptom onset. After multivariable adjustment, percentage of eGFR decreased from acute phase to follow-up was 8.30% (95% CI, 5.99-10.61) higher among AKI participants than those without AKI at acute phase. Participants with AKI had an odds ratio (OR) of 4.60 (95% CI, 2.10-10.08) for reduced renal function at follow-up. The percentage of eGFR decreased for participants with AKI stage 1, stage 2, and stage 3 was 6.02% (95% CI, 3.48-8.57), 15.99% (95% CI, 10.77-21.22), and 17.79% (95% CI, 9.14-26.43) higher compared with those without AKI, respectively.AKI at acute phase of COVID-19 was closely related to the longitudinal decline and post-acute status of kidney function at nearly one-year after symptom onset. Earlier and more intense follow-up strategies on kidney function management could be beneficial to COVID-19 survivors.Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS 2020-I2M-CoV19-005, 2018-I2M-1-003, and 2020-I2M-2-013); National Natural Science Foundation of China (82041011); National Key Research and Development Program of China (2018YFC1200102); Major Projects of National Science and Technology on New Drug Creation and Development of Pulmonary Tuberculosis (2020ZX09201001).

Geothermal is considered to be one of the most promising renewable sources for district heating. Ground source heat pump systems coupled with the coaxial deep borehole heat exchanger have been widely applied because of their high efficiency. However, in order to reduce the total energy consumption of the system, there are few studies on the optimization of the water flow rates in the coaxial deep borehole heat exchanger by time to meet the load change during the heating season. In this paper, an optimization method for the two flow rates which are set at two time periods respectively every day in the coaxial deep borehole heat exchanger during the operation of the ground source heat pump system is proposed. The application of the method is to determine the applicable flow rates when the temperature of the next day is predicted. Space heating of a building (located in Tianjin, China) is taken as a scenario, comparisons were carried out on system operation before and after optimization, which shows that the method has a good effect on the energy-saving operation of the system. After optimization, the total power consumption of the system is reduced, the total performance coefficient of the equipment as well as the temperature difference between the inlet and outlet of the underground heat exchanger is increased, and the trend of flow rates changes with time is the same as the indoor and outdoor temperature difference.

Data on the long-term trajectories of lung function are scarce in COVID-19 survivors.We re-analyzed the data from a prospective longitudinal cohort follow-up study of COVID-19 survivors over 2 years after infection. All participants were divided into scale 3, scale 4 and scale 5-6 groups according to seven-category ordinal scale. The changes of pulmonary function tests (PFTs), the Modified Medical Research Council (mMRC) Dyspnea Scale, 6-min walking test health-related quality of life (HRQoL) across the three serial follow-up visits were evaluated, and compared among three groups. We performed liner regression to determine potential factors that were associated with changes of PFTs and distance walked in 6 minutes (6MWD).In this study, 288 participants generally presented an improvement of PFTs parameters from 6 months to 1 year after infection. The scale 5-6 group displayed a significantly higher increase of PFTs compared with scale 3 and scale 4 groups (all p<0.0167), and corticosteroids therapy was identified as a protective factor for the PFTs improvement with a correlation coefficient of 2.730 (0.215-5.246) for forced vital capacity (FVC), 2.909 (0.383-5.436) for total lung capacity (TLC), and 3.299 (0.211-6.387) for diffusion capacity for carbon monoxide (DLco), respectively. From 1-year to 2-year follow-up, the PFTs parameters generally decreased, which was not observed to be associated with changes of 6MWD and HRQoL. Dyspnea (mMRC≥1) generally decreased over time (23.3% [61/262] for 6-month, 27.9% [67/240] for 1-year, 13.4% [35/261] for 2-year), and 6MWD increased continuously (500.0 m vs 505.0 m vs 525.0 m).Corticosteroids therapy during hospitalization was a protective factor for PFTs improvement from 6 months to 1 year. The relatively fast decline trend of PFTs from 1 year to 2 years needs to be paid attention and further validated in the future follow-up study.This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS 2021-I2M-1-048) and the National Key Research and Development Program of China (2021YFC0864700).

As a debilitating condition that can impact a whole spectrum of people and involve multi-organ systems, long COVID has aroused the most attention than ever. However, mechanisms of long COVID are not clearly understood, and underlying biomarkers that can affect the long-term consequences of COVID-19 are paramount to be identified.Participants for the current study were from a cohort study of COVID-19 survivors discharged from hospital between Jan 7, and May 29, 2020. We profiled the proteomic of plasma samples from hospitalised COVID-19 survivors at 6-month, 1-year, and 2-year after symptom onset and age and sex matched healthy controls. Fold-change of >2 or <0.5, and false-discovery rate adjusted P value of 0.05 were used to filter differentially expressed proteins (DEPs). In-genuity pathway analysis was performed to explore the down-stream effects in the dataset of significantly up- or down-regulated proteins. Proteins were integrated with long-term consequences of COVID-19 survivors to explore potential biomarkers of long COVID.The proteomic of 709 plasma samples from 181 COVID-19 survivors and 181 matched healthy controls was profiled. In both COVID-19 and control group, 114 (63%) were male. The results indicated four major recovery modes of biological processes. Pathways related to cell-matrix interactions and cytoskeletal remodeling and hypertrophic cardiomyopathy and dilated cardiomyopathy pathways recovered relatively earlier which was before 1-year after infection. Majority of immune response pathways, complement and coagulation cascade, and cholesterol metabolism returned to similar status of matched healthy controls later but before 2-year after infection. Fc receptor signaling pathway still did not return to status similar to healthy controls at 2-year follow-up. Pathways related to neuron generation and differentiation showed persistent suppression across 2-year after infection. Among 98 DEPs from the above pathways, evidence was found for association of 11 proteins with lung function recovery, with the associations consistent at two consecutive or all three follow-ups. These proteins were mainly enriched in complement and coagulation (COMP, PLG, SERPINE1, SRGN, COL1A1, FLNA, and APOE) and hypertrophic/dilated cardiomyopathy (TPM2, TPM1, and AGT) pathways. Two DEPs (APOA4 and LRP1) involved in both neuron and cholesterol pathways showed associations with smell disorder.The study findings provided molecular insights into potential mechanism of long COVID, and put forward biomarkers for more precise intervention to reduce burden of long COVID.National Natural Science Foundation of China; Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences; Clinical Research Operating Fund of Central High Level Hospitals; the Talent Program of the Chinese Academy of Medical Science; Training Program of the Big Science Strategy Plan; Ministry of Science and Technology of the People's Republic of China; New Cornerstone Science Foundation; Peking Union Medical College Education Foundation; Research Funds from Health@InnoHK Program.

Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1–3 and 7–8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4–6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.

A highly efficient iron-catalyzed approach to polysubstituted pyrroles has been developed through the [4C+1N] cyclization of 4-acetylenic ketones with primary amines, leading to the synthesis of a variety of tetra- and fully-substituted pyrroles as well as fused pyrrole derivatives in good to excellent yields.

According to the Third International Consensus Definition for Sepsis and Septic Shock, sepsis is a life-threatening organ dysfunction resulting from dysregulated host responses to infection. Epidemiological data about sepsis from the 2017 Global Burden of Diseases, Injuries and Risk Factor Study showed that the global burden of sepsis was greater than previously estimated. Bacteria have been shown to be the predominant pathogen of sepsis among patients with pathogens detected, while sepsis caused by viruses is underdiagnosed worldwide. The coronavirus disease that emerged in 2019 in China and now in many other countries has brought viral sepsis back into the vision of physicians and researchers worldwide. Although the current understanding of the pathophysiology of sepsis has improved, the differences between viral and bacterial sepsis at the level of pathophysiology are not well understood. Diagnosis methods that can broadly differentiate between bacterial and viral sepsis at the initial stage after the development of sepsis are limited. New treatments that can be applied at clinics for sepsis are scarce and this situation is not consistent with the growing understanding of pathophysiology. This review aims to give a brief summary of current knowledge of the epidemiology, pathophysiology, diagnosis and treatment of viral sepsis.

The possible effects of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) on COVID-19 disease severity have generated considerable debate. We performed a single-center, retrospective analysis of hospitalized adult COVID-19 patients in Wuhan, China, who had definite clinical outcome (dead or discharged) by February 15, 2020. Patients on anti-hypertensive treatment with or without ACEI/ARB were compared on their clinical characteristics and outcomes. The medical records from 702 patients were screened. Among the 101 patients with a history of hypertension and taking at least one anti-hypertensive medication, 40 patients were receiving ACEI/ARB as part of their regimen, and 61 patients were on antihypertensive medication other than ACEI/ARB. We observed no statistically significant differences in percentages of in-hospital mortality (28% vs. 34%, P = 0.46), ICU admission (20% vs. 28%, P = 0.37) or invasive mechanical ventilation (18% vs. 26%, P = 0.31) between patients with or without ACEI/ARB treatment. Further multivariable adjustment of age and gender did not provide evidence for a significant association between ACEI/ARB treatment and severe COVID-19 outcomes. Our findings confirm the lack of an association between chronic receipt of renin-angiotensin system antagonists and severe outcomes of COVID-19. Patients should continue previous anti-hypertensive therapy until further evidence is available.

Abstract Background The characteristics of neutralizing antibodies (NAbs) and antibody against major antigen proteins related to clinical outcomes in severe coronavirus disease 2019 (COVID-19) patients were still less known. Methods NAbs and antibodies targeting nucleocapsid (N), spike protein (S), and the receptor-binding domain (RBD) in longitudinal plasma samples from the LOTUS China trial were measured by microneutralization assay and enzyme-linked immunosorbent assay (ELISA). Viral load was determined by real-time reverse transcription polymerase chain reaction (RT-PCR). A total of 576 plasma and 576 throat swabs were collected from 191 COVID-19 patients. Antibody titers related to adverse outcome and clinical improvement were analyzed. Multivariable adjusted generalized linear mixed model for random effects were developed. Results After day 28 post symptoms onset, the rate of antibody positivity reached 100% for RBD-immunoglobulin M (IgM), 97.8% for S-IgM, 100% for N-immunoglobulin G (IgG), 100% for RBD-IgG, 91.1% for N-IgM, and 91.1% for NAbs. The NAbs titers increased over time in both survivors and nonsurvivors and correlated to IgG antibodies against N, S, and RBD, whereas its presence showed no statistical correlation with death. N-IgG (slope −2.11, 95% confidence interval [CI] −3.04 to −1.18, P &amp;lt; .0001), S-IgG (slope −2.44, 95% CI −3.35 to −1.54, P &amp;lt; .0001), and RBD-IgG (slope −1.43, 95% CI −1.98 to −.88, P &amp;lt; .0001) were negatively correlated with viral load. S-IgG titers were lower in nonsurvivors than survivors (P = .020) at week 4 after symptoms onset. Conclusions IgM and IgG against N, S, and RBD and NAbs developed in most severe COVID-19 patients and do not correlate clearly with clinical outcomes. The levels of IgG antibodies against N, S, and RBD were related to viral clearance.

<h2>Abstract</h2><h3>Objectives</h3> Use of corticosteroids is common in the treatment of coronavirus disease 2019, but clinical effectiveness is controversial. We aimed to investigate the association of corticosteroids therapy with clinical outcomes of hospitalized COVID-19 patients. <h3>Methods</h3> In this single-centre, retrospective cohort study, adult patients with confirmed coronavirus disease 2019 and dead or discharged between 29 December 2019 and 15 February 2020 were studied; 1:1 propensity score matchings were performed between patients with or without corticosteroid treatment. A multivariable COX proportional hazards model was used to estimate the association between corticosteroid treatment and in-hospital mortality by taking corticosteroids as a time-varying covariate. <h3>Results</h3> Among 646 patients, the in-hospital death rate was higher in 158 patients with corticosteroid administration (72/158, 45.6% vs. 56/488, 11.5%, p < 0.0001). After propensity score matching analysis, no significant differences were observed in in-hospital death between patients with and without corticosteroid treatment (47/124, 37.9% vs. 47/124, 37.9%, p 1.000). When patients received corticosteroids before they required nasal high-flow oxygen therapy or mechanical ventilation, the in-hospital death rate was lower than that in patients who were not administered corticosteroids (17/86, 19.8% vs. 26/86, 30.2%, log rank p 0.0102), whereas the time from admission to clinical improvement was longer (13 (IQR 10–17) days vs. 10 (IQR 8–13) days; p < 0.001). Using the Cox proportional hazards regression model accounting for time varying exposures in matched pairs, corticosteroid therapy was not associated with mortality difference (HR 0.98, 95% CI 0.93–1.03, p 0.4694). <h3>Discussion</h3> Corticosteroids use in COVID-19 patients may not be associated with in-hospital mortality.

<h2>Abstract</h2><h3>Background</h3> The pharmacokinetics and appropriate dose regimens of favipiravir are unknown in hospitalized influenza patients; such data are also needed to determine dosage selection for favipiravir trials in COVID-19. <h3>Methods</h3> In this dose-escalating study, favipiravir pharmacokinetics and tolerability were assessed in critically ill influenza patients. Participants received one of two dosing regimens; Japan licensed dose (1600 mg BID on day 1 and 600 mg BID on the following days) and the higher dose (1800 mg/800 mg BID) trialed in uncomplicated influenza. The primary pharmacokinetic endpoint was the proportion of patients with a minimum observed plasma trough concentration (C_trough) ≥20 mg/L at all measured time points after the second dose. <h3>Results</h3> Sixteen patients were enrolled into the low dose group and 19 patients into the high dose group of the study. Favipiravir C_trough decreased significantly over time in both groups (<i>p</i> <0.01). Relative to day 2 (48 hrs), concentrations were 91.7% and 90.3% lower in the 1600/600 mg group and 79.3% and 89.5% lower in the 1800/800 mg group at day 7 and 10, respectively. In contrast, oseltamivir concentrations did not change significantly over time. A 2-compartment disposition model with first-order absorption and elimination described the observed favipiravir concentration-time data well. Modeling demonstrated that less than 50% of patients achieved C_trough ≥20 mg/L for >80% of the duration of treatment of the two dose regimens evaluated (18.8% and 42.1% of patients for low and high dose regimen, respectively). Increasing the favipravir dosage predicted a higher proportion of patients reaching this threshold of 20 mg/L, suggesting that dosing regimens of ≥3600/2600 mg might be required for adequate concentrations. The two dosing regimens were well-tolerated in critical ill patients with influenza. <h3>Conclusion</h3> The two dosing regimens proposed for uncomplicated influenza did not achieve our pre-defined treatment threshold.

Background Cytokine storm observed in patients with severe Coronavirus Disease 2019 (COVID-19) contributes to poor clinical outcomes and increased mortality. Janus kinases (JAKs) are important mediators in the cytokine storm. Therefore, we conduct a living systematic review and meta-analysis of the literature investigating efficacy and safety of JAK inhibitors for patients with COVID-19. Methods Databases were searched up to December 1, 2021 for interventional and observational studies comparing JAK inhibitor treatment with concurrent control in patients with COVID-19. Efficacy and safety outcomes were evaluated by pooled risk ratio (RR). Results Of 3,170 records retrieved, 15 studies were eligible and 13 were evaluated in the meta-analysis ( n = 3,977). Based on data from three randomized controlled trials (RCTs), baricitinib treatment significantly decreased mortality by day 28 in hospitalized patients with COVID-19 (RR = 0.64, 95% CI 0.51–0.80) without increasing the incidence of adverse outcomes. In subgroup analysis, patients who required supplemental oxygen (RR = 0.62, 95% CI 0.41–0.95) or high-flow oxygen/non-invasive ventilation (RR = 0.59, 95% CI 0.42–0.85) at baseline benefited most. Pooled analysis of all eligible studies for JAK inhibitors (baricitinib, ruxolitinib, tofacitinib, and nezulcitinib) demonstrated a significant decrease in mortality (RR = 0.62, 95% CI 0.49–0.78) with no increase in the risk of adverse events. Conclusion Baricitinib probably decreases mortality in hospitalized adult patients with COVID-19, especially for patients who required supplemental oxygen or high-flow oxygen/non-invasive ventilation at baseline. The efficacy and safety of other JAK inhibitors, such as ruxolitinib, tofacitinib, and nezulcitinib, await more evidence. Systematic Review Registration https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021261414 , identifier: CRD42021261414.

The combined chemistry of silver and isocyanides is unique in performance, proved with the number of recent achievements. With the aim of triggering further research in this field, this review assembles and comprehensively summarizes the silver-based reactions of isocyanides. In this review, we especially emphasize the uniqueness of silver catalysis in the activation of isocyanides, the reaction selectivity, and the mechanistic description.

The geothermal energy is considered as one of the most promising renewable techniques for district heating. The deep coaxial borehole heat exchanger (BHE) is an economic and efficient measure for extracting the geothermal energy from underground, which attracts numerous attentions recently. However, the researches on the influences of the operation modes on the periodical heat extraction performances of the deep coaxial BHE are rare. In this paper, an unsteady numerical model of the dynamic operation of the coaxial borehole heat exchanger is established, based on the principles of energy conservation and mass conservation. The proposed model is verified using the operating data from the literature. Space heating of a school building (located at Tianjin, China) is taken as a scenario, and different operation modes during the heating period are simulated and analyzed. Comparisons were carried out on four operating modes: the continuous operation mode, “16+8 mode” (run for 16 h and stop for 8 h), “12+12 mode” and “8+16 mode”. The recovery of underground temperature distribution during the winter holiday was studied. Results show that, except for the continuous operation mode, the differences between the other three modes on the total extracted heat throughout the whole year and during the 24 h within the 90th day are unobvious. Under the “12+12 mode”, when the BHE reaches the stable level, the heat extraction power can be maintained at 301.6–528.1 kW in the first year, and the decline rate lower than 15% during the 20-year operation period. In addition, at 1 m from the center of the tube and at depths of 500 m, 1000 m, 1500 m, and 2000 m, the recovery rates of the underground temperature distribution relative to the initial temperature during the winter vacation are 7.35%, 13.78%, 16.57%, and 17.74%, respectively.

A novel iron-catalyzed aminolysis of β-carbonyl 1,3-dithianes with various amines including ammonia, primary and secondary amines, as well as hydrazine hydrate has been developed, leading to the synthesis of stereodefined β-enaminones and 3,4-disubstituted pyrazoles in good to high yields.

Stacking interactions between amino acids and bases are common in RNA–protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 3′ UTR (3′-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA–protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA–protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to “target” versus “off-target” interactions, and thus be an important consideration in the design of proteins with new specificities.

An atom-economic route to thiophenes and bithiophenes has been developed starting from the readily available gem-dialkylthio enynes. A range of functionalized thiophenes and bithiophenes, bearing a pendent vinylthio group, were obtained in good to high yields under mild conditions.

mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

The objective of this study was to investigate the difference in disease severity between influenza A and B among hospitalized adults using a novel ordinal scale and existing clinical outcome end points.A prospective, observational study was conducted over the 2016-2018 influenza seasons in a central hospital. The primary outcome was the rate of clinical improvement, defined as a decline of 2 categories from admission on a 7-category ordinal scale that ranges from 1 (discharged with normal activity) to 7 (death), or hospital discharge up to day 28.In total, 574 eligible patients were enrolled, including 369 (64.3%) influenza A cases and 205 (35.7%) influenza B cases. The proportion of patients with a worse ordinal scale at admission was higher in influenza A than influenza B (P = .0005). Clinical improvement up to 28 days occurred in 82.4% of patients with influenza A and 90.7% of patients with influenza B (P = .0067). The Cox model indicated that influenza B patients had a higher clinical improvement probability than influenza A cases (adjusted hazard ratio [HR], 1.266; 95% confidence interval [CI], 1.019-1.573; P = .0335). A similar pattern was observed in weaning oxygen supplement (adjusted HR, 1.285; 95% CI, 1.030-1.603; P = .0261). In-hospital mortality for influenza A was marginally higher than influenza B (11.4% vs 6.8%; P = .0782).Our findings indicated that hospitalized patients with influenza A were more ill and had delayed clinical improvement compared with those with influenza B virus infection.

Abstract Background Respiratory syncytial virus (RSV) is an important cause of medically attended acute respiratory illnesses in older adults but awareness of the relevance of RSV in older people remains lower than that of influenza, which exhibits similar clinical characteristics to those of RSV. Objectives This study was performed to assess the clinical significance of RSV in respiratory samples from hospitalized adults. Methods Characteristics and outcomes in adults (≥18 years) hospitalized for RSV infection (n = 51) were compared with a cohort hospitalized for influenza A infection (n = 279) in a single‐center retrospective cohort study in Beijing, China. Results Respiratory syncytial virus patients were slightly older, with no significant differences in underlying chronic conditions. Lower respiratory tract infection and cardiovascular complications were more frequent ( P &lt; .05) in RSV patients. Rates of mortality in the RSV cohorts were significantly higher within 30 days (13.7% vs 5.0%, P = .019) and 60 days (17.6% vs 7.5%, P = .021). Bacterial co‐infection in respiratory samples was associated with reduced survival among RSV patients (log rank, P = .013). Conclusions Respiratory syncytial virus is a common cause of serious illness among hospitalized adults in China with greater mortality than influenza A. Increased awareness and the availability of antiviral agents might increase the scope for successful management.

Abstract The purpose of this study is to explore whether uric acid (UA) can independently act as a prognostic factor and critical marker of the 2019 novel corona virus disease (COVID-19). A multicenter, retrospective, and observational study including 540 patients with confirmed COVID-19 was carried out at four designated hospitals in Wuhan. Demographic, clinical, laboratory data were collected and analyzed. The primary end point was in-hospital death of patients with COVID-19. The concentration of admission UA ( ad UA) and the lowest concentration of uric acid during hospitalization ( low UA) in the dead patients were significantly lower than those in the survivors. Multivariate logistic regression analysis showed the concentration of low UA (OR 0.986, 95% CI 0.980–0.992, p &lt; 0.001) was able to independently predict the risk of in-hospital death. The mean survival time in the low-level group of low UA was significantly lower than other groups. When low UA was ≤ 166 µmol/L, the sensitivity and specificity in predicting hospital short-term mortality were 76.9%, (95% CI 68.5–85.1%) and 74.9% (95% CI 70.3–78.9%). This retrospective study determined that the lowest concentration of UA during hospitalization can be used as a prognostic indicator and a marker of disease severity in severe patients with COVID-19.

BackgroundThe Xpert Xpress Flu/RSV assay is released by FDA for rapid detection of influenza A (FluA), influenza B (FluB), and respiratory syncytial virus (RSV). This study aimed to evaluate its clinical performance in comparison to that of the RT-PCR assay cleared by China FDA (CFDA-PCR).MethodsNasopharyngeal specimens were collected from patients and tested by the two assays side by side. Discordant results were tested with a laboratory-developed real-time PCR for resolution. Viral load in the sample was quantified with a droplet digital PCR.ResultsA total of 658 specimens were involved and gave 94.7%–99.1% agreement between the two assays. The Xpert assay showed higher sensitivity for FluA (100% vs. 89.8%) and FluB detection (100% vs. 95.3%), and also higher accuracy (98.9% vs. 95.7%) for FluA than the CDFA-PCR. The positive and negative predictive values (NPV) for the three viruses ranged from 90.5% to 100% in the two assays, with higher NPV for FluA and FluB in Xpert assay. Moreover, the Xpert Ct values showed a linear correlation with virus titer in specimens tested.ConclusionOverall, the Xpert assay is a reliable and sensitive tool for the detection of FluA, FluB and RSV in our clinical settings.

Since the first reaction involved ylides was reported, they have been an important research direction of organic chemistry.Among them, iodonium ylides attracted considerable attentions in organic synthesis due to their unique reactivities as both iodine source and ylides.The preparation methods and structural properties of iodonium ylides are generally elaborated.Then, the reactivities of iodonium ylides are reviewed in detal, including the application of iodonium ylides as a carbene precursor in insertion reactions, cyclopropane reactions, and their development research in cycloaddition reactions, rearrangement reactions and halogenation reaction.

The solar-assisted heat pump (SAHP) is considered to be one of the efficient and promising clean heating technologies, while the research on its operation strategy is insufficient. In this study, a novel triangular solar air collector assisted air source heat pump (TSAHP) for rural residence heating is presented. Three working modes of the TSAHP including preheating, series and parallel modes were investigated, and corresponding mathematical models were established and verified. The effects of solar irradiance, outdoor temperature, indoor temperature, south wall covered area of the TSAC and the heating load on the performances of the three working modes were analyzed. The distribution diagram of the optimal working mode was designed, and the equations that define the boundaries between operating modes were fitted. Finally, the performances of the TSAHP under typical meteorological parameters were compared and analyzed based on the modified boundary equations. Results indicate that the TSAHP can sufficiently utilize solar energy. With the increase of solar irradiance, the optimal working mode is shifted from the preheating to series and parallel. Compared with the traditional air source heat pump system, the COP of the TSAHP under the optimal working modes could be increased by 64.4%.

The ice storage system is one of the most promising techniques for flexibility enhancement of building cooling load. In this paper, the performances of smooth-tube and corrugated-tube heat exchangers for ice storage are compared by experiments and numerical simulation. The heat transfer process of the ice storage and melting within the heat exchangers is numerically analyzed used Python. The fast one-dimensional numerical model is verified by the experimental results. The temperature variation of the heat transfer fluid (HTF) in the corrugated tube is analyzed and compared with that in the smooth tube. The results show that under the same conditions, the ice storage duration of the corrugated tube is shortened by 25% and the melting duration is shortened by 16% compared with those of the smooth tube. During the ice storage process, the temperature difference between the inlet and outlet of the heat transfer fluid in the corrugated tube is 0.4 °C less than that of the smooth tube on average. The temperature change rate is 30% faster than that of the smooth tube. During the melting process, the temperature difference between the inlet and outlet of the heat transfer fluid in the corrugated tube is 0.1 °C higher than that of the smooth tube on average, and the temperature change rate of corrugated-tube heat exchanger is 2.5 times and 9 times faster than that of the smooth tube during the ice-storage and melting process. The corrugated-tube heat exchanger has superior performances in ice storage system in the comparison with the smooth-tube heat exchanger.

In the presence of carbon tetrabromide, a variety of dithiocarbamates, xanthates, dithioesters, and thioethers were prepared in one pot by reacting the corresponding dithioic acids or thiols with active methylene compounds/indole derivatives under mild conditions. The formation of a sulfenyl bromide intermediate is proposed as the key step, which initiates the C-S bond formation.

The global change in protein abundance in colorectal cancer (CRC) and its contribution to tumorigenesis have not been comprehensively analyzed. In this study, we conducted a comprehensive proteomic analysis of paired tumors and adjacent tissues (AT) using high-resolution Fourier-transform mass spectrometry and a novel algorithm of quantitative pathway analysis. 12380 proteins were identified and 740 proteins that presented a 4-fold change were considered a CRC proteomic signature. A significant pattern of changes in protein abundance was uncovered which consisted of an imbalance in protein abundance of inhibitory and activating regulators in key signal pathways, a significant elevation of proteins in chromatin modification, gene expression and DNA replication and damage repair, and a decreased expression of proteins responsible for core extracellular matrix architectures. Specifically, based on the relative abundance, we identified a panel of 11 proteins to distinguish CRC from AT. The protein that showed the greatest degree of overexpression in CRC compared to AT was Dipeptidase 1 (DPEP1). Knockdown of DPEP1 in SW480 and HCT116 cells significantly increased cell apoptosis and attenuated cell proliferation and invasion. Together, our results show one of largest dataset in CRC proteomic research and provide a molecular link from genomic abnormalities to the tumor phenotype.

Evidence concerning the efficacy and safety of extracorporeal membrane oxygenation (ECMO) in patients with influenza A (H7N9) has been was limited to case reports. Our study is aimed to investigate the current application, efficacy and safety of ECMO in for severe H7N9 pneumonia-associated acute respiratory distress syndrome (ARDS) in the Chinese population. A multicentre retrospective cohort study was conducted at 20 hospitals that admitted patients with avian influenza A (H7N9) viral pneumonia patients’ admission from 9 provinces in China between October 1, 2016, and March 1, 2017. Data from the National Health and Family Planning Commission of China, including general conditions, outcomes and ECMO management, were analysed. Then, successfully weaned and unsuccessfully weaned groups were compared. A total of 35 patients, aged 57 ± 1 years, were analysed; 65.7% of patients were male with 63% mortality. All patients underwent invasive positive pressure ventilation (IPPV), and rescue ventilation strategies were implemented for 23 cases (65.7%) with an average IPPV duration of 5 ± 1 d, PaO2/FiO2 of 78 ± 23 mmHg, tidal volume (VT) of 439 ± 61 ml and plateau pressure (Pplat) of 29 ± 8 cmH2O pre-ECMO. After 48 h on ECMO, PaO2 improved from 56 ± 21 mmHg to 90 ± 24 mmHg and PaCO2 declined from 52 ± 24 mmHg to 38 ± 24 mmHg. Haemorrhage, ventilator-associated pneumonia (VAP) and barotrauma occurred in 45.7%, 60% and 8.6% of patients, respectively. Compared with successfully weaned patients (n = 14), the 21 unsuccessfully weaned patients had a longer duration of IPPV pre-ECMO (6 ± 4 d vs. 2 ± 1 d, P < 0.01) as well as a higher Pplat (25 ± 5 cmH2O vs. 21 ± 3 cmH2O, P < 0.05) and VT (343 ± 96 ml vs. 246 ± 93 ml, P < 0.05) after 48 h on ECMO support. Furthermore, the unsuccessfully weaned group had a higher mortality (100% vs. 7.1%, P < 0.01) with more haemorrhage (77.3% vs. 28.6%, P < 0.01). ECMO is effective at improving oxygenation and ventilation of patients with avian influenza A (H7N9) induced severe ARDS. Early initiation of ECMO with appropriate IPPV settings and anticoagulation strategies are necessary to reduce complications.

Abstract Glioblastoma (GBM) is the most common and malignant Grade IV primary craniocerebral tumor caused by glial cell carcinogenesis with an extremely poor median survival of 12–18 months. The current standard treatments for GBM, including surgical resection followed by chemotherapy and radiotherapy, fail to substantially prolong survival outcomes. Adeno-associated virus (AAV)-mediated gene therapy has recently attracted considerable interest because of its relatively low cytotoxicity, poor immunogenicity, broad tissue tropism, and long-term stable transgene expression. Furthermore, a range of gene therapy trials using AAV as vehicles are being investigated to thwart deadly GBM in mice models. At present, AAV is delivered to the brain by local injection, intracerebroventricular (ICV) injection, or systematic injection to treat experimental GBM mice model. In this review, we summarized the experimental trials of AAV-based gene therapy as GBM treatment and compared the advantages and disadvantages of different AAV injection approaches. We systematically introduced the prospect of the systematic injection of AAV as an approach for AAV-based gene therapy for GBM.

Long non-coding RNAs (lncRNAs) have critical functions in tumorigenesis and progression of colorectal cancer (CRC). The role of lncRNA COL4A2-AS1 (COL4A2-AS1) lacks system investigation. The current study comprehensively analyzed the expression, biological functions, and mechanism of COL4A2-AS1 in CRC through performing real-time quantitative PCR (RT-qPCR), Western blot, cell transfection, cell colony assay, MTT assay, flow cytometry and dual-luciferase reporter system assays. A xenograft model of CRC was constructed to further verify the function of COL4A2-AS1 in CRC progression in vivo. The data revealed an upregulated expression of COL4A2-AS1 in CRC tissues and cell lines than paired adjacent tissues and normal cell line. Silencing COL4A2-AS1 inhibited proliferation, aerobic glycolysis, and promoted apoptosis of CRC cells in vivo and in vitro. However, overexpression of COL4A2-AS1 significantly promoted CRC cell proliferation and aerobic glycolysis. In CRC cells, miR-20b-5p was sponged by COL4A2-AS1 and hypoxia-inducible factor 1 alpha subunit (HIF1A). Restoration of HIF1A expression reversed the inhibitory effects of silencing COL4A2-AS1 on aerobic glycolysis and proliferation of CRC cells. The current findings showed that COL4A2-AS1 promoted the proliferation, and aerobic glycolysis of CRC cells potentially through modulating the miR-20b-5p/HIF1A axis.

Background The diagnosis of bacterial pathogens in lower respiratory tract infections (LRI) using conventional culture methods remains challenging and time-consuming. Objectives To evaluate the clinical performance of a rapid nanopore-sequencing based metagenomics test for diagnosis of bacterial pathogens in common LRIs through a large-scale prospective study. Methods We enrolled 292 hospitalized patients suspected to have LRIs between November 2018 and June 2019 in a single-center, prospective cohort study. Rapid clinical metagenomics test was performed on-site, and the results were compared with those of routine microbiology tests. Results 171 bronchoalveolar lavage fluid (BAL) and 121 sputum samples were collected from patients with six kinds of LRIs. The turnaround time (from sample registration to result) for the rapid metagenomics test was 6.4 ± 1.4 hours, compared to 94.8 ± 34.9 hours for routine culture. Compared with culture and real-time PCR validation tests, rapid metagenomics achieved 96.6% sensitivity and 88.0% specificity and identified pathogens in 63 out of 161 (39.1%) culture-negative samples. Correlation between enriched anaerobes and lung abscess was observed by Gene Set Enrichment Analysis. Moreover, 38 anaerobic species failed in culture was identified by metagenomics sequencing. The hypothetical impact of metagenomics test proposed antibiotic de-escalation in 34 patients compared to 1 using routine culture. Conclusions Rapid clinical metagenomics test improved pathogen detection yield in the diagnosis of LRI. Empirical antimicrobial therapy could be de-escalated if rapid metagenomics test results were hypothetically applied to clinical management.

Antibodies have pivotal roles in the immune response to infection. Laboratory-created SARS-CoV-2 antibody treatment is an emerging approach to treat COVID-19. Early treatment with these antibodies can reduce COVID-19-related hospitalisation or death in patients at high risk.1Weinreich DM Sivapalasingam S Norton T et al.REGN-COV2, a neutralizing antibody cocktail, in outpatients with COVID-19.N Engl J Med. 2021; 384: 238-251Crossref PubMed Scopus (1120) Google Scholar, 2Gupta A Gonzalez-Rojas Y Juarez E et al.Early treatment for COVID-19 with SARS-CoV-2 neutralizing antibody sotrovimab.N Engl J Med. 2021; 385: 1941-1950Crossref PubMed Scopus (616) Google Scholar, 3Dougan M Nirula A Azizad M et al.Bamlanivimab plus etesevimab in mild or moderate COVID-19.N Engl J Med. 2021; 385: 1382-1392Crossref PubMed Scopus (426) Google Scholar These antibodies can also provide targeted prophylaxis for individuals at high risk, and interrupt transmission of viruses in populations.4Marston HD Paules CI Fauci AS Monoclonal antibodies for emerging infectious diseases—borrowing from history.N Engl J Med. 2018; 378: 1469-1472Crossref PubMed Scopus (99) Google Scholar A major concern of SARS-CoV-2 antibodies is the limited commercial value compared with current small-molecule drugs, such as nirmatrelvir plus ritonavir (Paxlovid) and molnupiravir. Small-molecule drugs (typically 0·1–0·6 kilodalton) can penetrate the cell membrane and bind intracellular targets; they are generally more stable than biological drugs and can be administered orally. Therefore, the target population that would benefit from SARS-CoV-2 antibodies rather than small-molecule drugs needs to be identified. Currently, SARS-CoV-2 antibody treatment might be effective in immunocompromised patients with COVID-19.5Denkinger CM Janssen M Schäkel U et al.Anti-SARS-CoV-2 antibody-containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19: a randomized clinical trial.Nat Cancer. 2023; 4: 96-107PubMed Google Scholar However, large phase 3 trials are difficult to conduct to establish the efficacy of SARS-CoV-2 antibody treatment in the highest-risk patients, who have negligible ability to produce endogenous antibodies. Examples of such patients include those with haematological malignancies treated with anti-CD20 monoclonal antibodies (mAbs) or chimeric antigen receptor T-cell immunotherapy. For these patients, systemic administration would be more helpful than inhalation due to their almost non-existent humoral immunity. Another promising use of SARS-CoV-2 antibodies is in prophylactic treatment for patients at high risk. The necessary condition is that the antibodies require an extended half-life to be effective.6Abraham J Monoclonal antibodies with extended half-life to prevent Covid-19.N Engl J Med. 2022; 386: 2236-2238Crossref PubMed Scopus (2) Google Scholar It is reasonable to position the objective of the phase 1/3 trial of AZD3152 (NCT05648110), a combination of two mAbs (AZD1061 and AZD3152), as evaluating its safety and neutralising activity for pre-exposure prophylaxis of COVID-19. However, theoretically, inhaling the antibodies might not provide long-lasting exposure in the lungs. Therefore, the use of the inhalation route for antibodies needs to be explored for its potential value and scenarios for SARS-CoV-2 infection, because it might also be helpful for other infectious diseases. The evasion of mAb-induced protection to all SARS-CoV-2 antibodies is the biggest threat because of the evolution of the spike protein of new SARS-CoV-2 variants.7Cox M Peacock TP Harvey WT et al.SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies.Nat Rev Microbiol. 2023; 21: 112-124Crossref PubMed Scopus (61) Google Scholar At the time of writing, the omicron variant (BA.5) has evaded almost all available mAb-based drugs.8Wang Q Iketani S Li Z et al.Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants.Cell. 2023; 186: 279-286Summary Full Text Full Text PDF PubMed Scopus (226) Google Scholar Efforts are still being coordinated to quickly identify and develop better antibodies.9Chen Y Zhao X Zhou H Zhu H Jiang S Wang P Broadly neutralizing antibodies to SARS-CoV-2 and other human coronaviruses.Nat Rev Immunol. 2023; 23: 189-199Crossref PubMed Scopus (47) Google Scholar Maranda and colleagues10Maranda B Labbé SM Lurquin M et al.Safety and efficacy of inhaled IBIO123 for mild-to-moderate COVID-19: a randomised, double-blind, dose-ascending, placebo-controlled, phase 1/2 trial.Lancet Infect Dis. 2023; (published online Aug 21.)https://doi.org/10.1016/S1473-3099(23)00393-6Google Scholar did a phase 1/2 trial to test the safety and explore the efficacy of IBIO123. This treatment consists of a fully human, recombinant, monoclonal IgG in a mixture of two antibodies binding to the S1 subunit and one antibody binding to the S2 subunit of the receptor-binding domain. IBIO123 showed neutralisation potency against SARS-CoV-2 from the original strain isolated in Wuhan, China, up to XBB1.5. The authors raised a hypothesis that direct delivery of neutralising antibodies via inhalation might provide additional respiratory clinical benefits. Thus, in this phase 1/2 trial, IBIO123 and a placebo were administered via oral inhalation to outpatients with COVID-19. The main findings of the study indicate that oral inhalation of IBIO123 did not result in more adverse events or serious adverse events than the placebo. The results also showed that the proportion of participants with respiratory symptom resolution on day 8 was 33 (41%) of 81 in the IBIO123 group, compared with five (17%) of 29 in the placebo group (p=0·024) in the overall population. However, no significant reduction in viral load was observed on day 5. The study showed the safety of inhaling antibodies. However, several key questions remain unanswered, including whether inhalation of antibodies is more beneficial than systemic administration, how inhalation of antibodies neutralises SARS-CoV-2 in other organs, how antibody exposure should be evaluated in the lungs and plasma, and how the optimal dosage of antibody could be identified in phase 1 trials. The extended development time required for new small-molecule drugs makes it challenging for them to address emerging infectious diseases quickly. Although SARS-CoV-2 antibodies face the aforementioned challenges, the rapid development of mAbs is poised to contribute more substantially in the future. The insights gained from antibodies for SARS-CoV-2 will probably be applicable to the creation of antibodies for other infectious diseases too. We declare no competing interests. Safety and efficacy of inhaled IBIO123 for mild-to-moderate COVID-19: a randomised, double-blind, dose-ascending, placebo-controlled, phase 1/2 trialInhalation of IBIO123 was safe. Despite the lack of significant reduction of viral load at day 5, treatment with IBIO123 resulted in a higher proportion of participants with complete resolution of respiratory symptoms at day 6. This study supports further clinical research on inhaled monoclonal antibodies in COVID-19 and respiratory diseases in general. Full-Text PDF

Glycosylasparaginase belongs to a family of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from single-chain protein precursors. Previously, based on a precursor structure paused at pre-autoproteolysis stage by a reversible inhibitor (glycine), we proposed a mechanism of intramolecular autoproteolysis. A key structural feature, a highly strained conformation at the scissile peptide bond, had been identified and was hypothesized to be critical for driving autoproteolysis through an N–O acyl shift. To examine this “twist-and-break” hypothesis, we report here a 1. 9-Å-resolution structure of an autoproteolysis-active precursor (a T152C mutant) that is free of inhibitor or ligand and is poised to undergo autoproteolysis. The current crystallographic study has provided direct evidence for the natural conformation of the glycosylasparaginase autocatalytic site without influence from any inhibitor or ligand. This finding has confirmed our previous proposal that conformational strain is an intrinsic feature of an active precursor.

Objective: The objective of the study was to determine whether statin use could reduce the risk of the incidence or progression of osteoarthritis (OA).Methods: The PubMed, Embase, and Cochrane databases were systematically searched for observational studies on the association between statin use and OA.ORs and 95% CIs were directly retrieved or calculated.The Newcastle-Ottawa quality assessment scale was used for study quality assessment.Subgroup analysis, sensitivity analysis, and publication bias were conducted using Stata software.Results: A total of 11 studies (679807 participants) were identified from the systematic literature search.No significant association between statin use and incidence (OR ¼ 1.010; 95% CI: 0.968 to 1.055; P ¼ 0.638) or progression (OR ¼ 1.076; 95% CI: 0.824 to 1.405; P ¼ 0.589) of OA was found in our metaanalysis.The meta-analysis according to the symptomatic or radiological OA also found no significant association between statin use and OA.The subgroup analysis showed that atorvastatin (OR ¼ 0.953; 95% CI: 0.911 to 0.998; P ¼ 0.041) and rosuvastatin (OR ¼ 1.180; 95% CI: 1.122 to 1.241; P < 0.0001) had opposite effects on OA.The results of the analysis according to the joint site, interval, and statin dose were all not significant.Conclusions: Statin use may not be associated with a lower risk of incidence and progression of OA, regardless of joint site.The opposite effects of atorvastatin and rosuvastatin were detected in OA.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a great threat to public health worldwide. Approximately 5% of patients are complicated with critical illness, with some of them experiencing multi-organ dysfunction and even death.1,2

Influenza A virus still represents a noticeable epidemic risk to international public health at present, despite the extensive use of vaccines and anti-viral drugs. In the fight against pathogens, the immune defence lines consisting of diverse lymphocytes are indispensable for humans. However, the role of virus infection of lymphocytes and subsequent abnormal immune cell death remains to be explored. Different T cell subpopulations have distinct characterizations and functions, and we reveal the high heterogeneity of susceptibility to viral infection and biological responses such as apoptosis in various CD4+ T and CD8+ T cell subsets through single-cell transcriptome analyses. Effector memory CD8+ T cells (CD8+ TEM) that mediate protective memory are identified as the most susceptible subset to pandemic influenza A virus infection among primary human T cells. Non-productive infection is established in CD8+ TEM and naïve CD8+ T cells, which indicate the mechanism of intracellular antiviral activities for inhibition of virus replication such as abnormal viral splicing efficiency, incomplete life cycles and up-regulation of interferon-stimulated genes in human T cells. These findings provide insights into understanding lymphopenia and the infectious mechanisms of pandemic influenza A virus and broad immune host–pathogen interactional atlas in primary human T cells.

A photocycloaddition reaction of ethyl 1,4-diaryl-1,4-dihydropyridine-3-carboxylate for the construction of 3,9-diazatetraasteranes (P1) and 3,9-diazatetracyclododecanes (P2) is reported for the first time. The types of reaction product clearly differ with solvent, regardless of the irradiation wavelength. The difference in P1 and P2 lies in the second step of the intramolecular [2 + 2] photocyclization. In order to further investigate this phenomenon and gain a deeper understanding of the photochemical behavior of 1,4-dihydropyridines, DFT and TDDFT theoretical calculations are performed. The results provide a good explanation for the formation of 3,9-diazatetraasteranes and 3,9-diazatetracyclododecanes.

Abstract In recent years, the silver‐catalyzed and silver‐promoted isocyanide reactions have attracted much attention, due to its efficiency in the formation of diverse new bonds and good reaction selectivity. A series of highly useful linear or cyclic compounds have been constructed. In this review, the recent progress in this field is described in the sequence of synthesis of five‐membered heterocyclic compounds, synthesis of six‐membered heterocyclic compounds, synthesis of fused cyclic compounds as well as synthesis of linear nitrogen‐containing compounds.

Corticosteroids have beneficial effects in improving outcomes in hospitalized patients with severe COVID-19 by suppressing excessive immune responses. However, the effect of corticosteroids on the humoral and T-cell responses of survivors of COVID-19 1 year after infection remains uncertain because it relates to the extent of immediate, antigen-specific defense provided by protective memory.What is the effect of corticosteroids on long-term humoral and T-cell immune responses?In this retrospective cohort study conducted at a single center, we analyzed data from a cohort who had survived COVID-19 to compare the 1-year seropositivity and titer changes in neutralizing antibodies (NAbs) and SARS-CoV-2-specific antibodies. Additionally, we evaluated the magnitude and rate of SARS-CoV-2-specific T-cell response in individuals who received corticosteroids during hospitalization and those who did not.Our findings indicated that corticosteroids do not statistically influence the kinetics or seropositive rate of NAbs against the Wuhan strain of SARS-CoV-2 from 6 months to 1 year. However, subgroup analysis revealed a numerical increase of absolute NAbs titers, from 20.0 to 28.2, in categories where long-term (> 15 days) and high-dose (> 560 mg) corticosteroids are administered. Similarly, corticosteroids showed no significant effect on nucleoprotein and receptor-binding domain IgG at 1 year, except for spike protein IgG (β, 0.08; 95% CI, 0.04-0.12), which demonstrated a delayed decline of titers. Regarding T-cell immunity, corticosteroids did not affect the rate or magnitude of T-cell responses significantly. However, functional assessment of memory T cells revealed higher interferon-γ responses in CD4 (β, 0.61; 95% CI, 0.10-1.12) and CD8 (β, 0.63; 95% CI, 0.11-1.15) memory T cells in the corticosteroids group at 1 year.Based on our findings, short-term and low-dose corticosteroid therapy during hospitalization does not have a significant effect on long-term humoral kinetics or the magnitude and rate of memory T-cell responses to SARS-CoV-2 antigens. However, the potential harmful effects of long-term and high-dose corticosteroid use on memory immune responses require further investigation.

The impact of corticosteroids on humoral responses in coronavirus disease 2019 (COVID-19) survivors during the acute phase and subsequent 6-month period remains unknown. This study aimed to determine how the use of corticosteroids influences the initiation and duration of humoral responses in COVID-19 survivors 6 months after infection onset. We used kinetic antibody data from the lopinavir–ritonavir trial conducted at Jin Yin-Tan Hospital in January 2020, which involved adults hospitalized with severe COVID-19 (LOTUS, ChiCTR2000029308). Antibody samples were collected from 192 patients during hospitalization, and kinetic antibodies were monitored at all available time points after recruitment. Additionally, plasma samples were collected from 101 COVID-19 survivors for comprehensive humoral immune measurement at the half-year follow-up visit. The main focus was comparing the humoral responses between patients treated with systemic corticosteroid therapy and the non-corticosteroid group. From illness onset to day 30, the median antibody titre areas under the receiver operating characteristic curve (AUCs) of nucleoprotein (N), spike protein (S), and receptor-binding domain (RBD) immunoglobulin G (IgG) were significantly lower in the corticosteroids group. The AUCs of N-, S-, and RBD-IgM as well as neutralizing antibodies (NAbs) were numerically lower in the corticosteroids group compared with the non-corticosteroid group. However, peak titres of N, S, RBD-IgM and -IgG and NAbs were not influenced by corticosteroids. During 6-month follow-up, we observed a delayed decline for most binding antibodies, except N-IgM (β −0.05, 95% CI [−0.10, 0.00]) in the corticosteroids group, though not reaching statistical significance. No significant difference was observed for NAbs. However, for the half-year seropositive rate, corticosteroids significantly accelerated the decay of IgA and IgM but made no difference to N-, S-, and RBD-IgG or NAbs. Additionally, corticosteroids group showed a trend towards delayed viral clearance compared with the non-corticosteroid group, but the results were not statistically significant (adjusted hazard ratio 0.71 95% CI 0.50–1.00; P = 0.0508). Our findings suggested that corticosteroid therapy was associated with impaired initiation of the antibody response but this did not compromise the peak titres of binding and neutralizing antibodies. Throughout the decay phase, from the acute phase to the half-year follow-up visit, short-term and low-dose corticosteroids did not significantly affect humoral responses, except for accelerating the waning of short-lived antibodies.

Abnormal changes of monocytes have been observed in acute COVID-19, whereas associations of monocyte count with long COVID were not sufficiently elucidated.A cohort study was conducted among COVID-19 survivors discharged from hospital. The primary outcomes were core symptoms of long COVID, distance walked in 6 min, and lung function, and the secondary outcomes were health-related quality of life and healthcare use after discharge. Latent variable mixture modeling was used to classify individuals into groups with similar trajectory of monocyte count from discharge to 2-year after symptom onset. Multivariable adjusted generalized linear regression models and logistic regression models were used to estimate the associations of monocyte count trajectories and monocyte count at discharge with outcomes.In total, 1389 study participants were included in this study. Two monocyte count trajectories including high to normal high and normal trajectory were identified. After multivariable adjustment, participants in high to normal high trajectory group had an odds ratio (OR) of 2.52 (95% CI, 1.44-4.42) for smell disorder, 2.27 (1.27-4.04) for 6-min walking distance less than lower limit of normal range, 2.45 (1.08-5.57) for total lung capacity (TLC) < 80% of predicted, 3.37 (1.16-9.76) for personal care problem, and 1.70 (1.12-2.58) for rehospitalization after discharge at 2-year follow-up compared with those in normal trajectory group. Monocyte count at discharge showed similar results, which was associated with smell disorder, TLC < 80% of predicted, diffusion impairment, and rehospitalization.Monocyte count may serve as an easily accessible marker for long-term management of people recovering from COVID-19.

Abstract The immune mechanisms of long coronavirus disease 2019 (COVID) are not yet fully understood. We aimed to investigate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific memory immune responses in discharged COVID-19 patients with and without long COVID symptoms. In this cross-sectional study, we included 1041 hospitalized COVID-19 patients with the original virus strain in Wuhan (China) 12 months after initial infection. We simultaneously conducted a questionnaire survey and collected peripheral blood samples from the participants. Based on the presence or absence of long COVID symptoms during the follow-up period, we divided the patients into 2 groups: a long COVID group comprising 480 individuals and a convalescent group comprising 561 individuals. Both groups underwent virus-specific immunological analyses, including enzyme-linked immunosorbent assay, interferon-γ-enzyme-linked immune absorbent spot, and intracellular cytokine staining. At 12 months after infection, 98.5% (1026/1041) of the patients were found to be seropositive and 93.3% (70/75) had detectable SARS-CoV-2-specific memory T cells. The long COVID group had significantly higher levels of receptor binding domain (RBD)–immunoglobulin G (IgG) levels, presented as OD450 values, than the convalescent controls (0.40 ± 0.22 vs 0.37 ± 0.20; P = .022). The magnitude of SARS-CoV-2-specific T-cell responses did not differ significantly between groups, nor did the secretion function of the memory T cells. We did not observe a significant correlation between SARS-CoV-2-IgG and magnitude of memory T cells. This study revealed that long COVID patients had significantly higher levels of RBD-IgG antibodies when compared with convalescent controls. Nevertheless, we did not observe coordinated SARS-CoV-2-specific cellular immunity. As there may be multiple potential causes of long COVID, it is imperative to avoid adopting a “one-size-fits-all” approach to future treatment modalities.

Sanhan Huashi granules (SHG) demonstrated therapeutic effects against coronavirus disease 2019 (COVID-19) in observational studies. In order to compare the effectiveness and safety of SHG and nirmatrelvir–ritonavir in treating adults with mild-to-moderate COVID-19, we conducted a randomized, active-controlled, open-label, multi-center trial conducted between February and July in 2023. The patients were randomized in a 1:1 ratio to the SHG group and the nirmatrelvir–ritonavir group. A total of 400 participants were randomized, among which 200 participants ultimately received SHG and 198 received nirmatrelvir–ritonavir. The primary outcome was time to sustained clinical recovery through day 28. SHG significantly shortened the median time to sustained clinical recovery compared to nirmatrelvir–ritonavir (6.0 [95% CI, 5.0 to 6.0] vs. 8.0 [CI,6.0 to 9.0] days; P = 0.001), particularly for individual symptoms including fever, sore throat, cough and fatigue. No participants in either group died and incidence of severe COVID-19 showed no difference between two groups. Participants who received nirmatrelvir–ritonavir demonstrated a higher rate of virus clearance on day 5 compared to those received SHG (46.4% [CI, 39.1 to 53.7] vs. 65.6% [CI, 58.3 to 72.4]; P < 0.001). Most adverse events were mild in both groups. In summary, SHG was superior to nirmatrelvir–ritonavir in shortening the time to sustained clinical recovery in participants with mild-to-moderate COVID-19, despite a lower virus clearance rate observed after 5 days of treatment (Chinese Clinical Trial Registry Identifier: ChiCTR2300067872).

<h2>Summary</h2><h3>Background</h3> Growing evidence suggests that symptoms associated with post-COVID-19 condition (also known as long COVID) can affect multiple organs and systems in the human body, but their association with viral persistence is not clear. The aim of this study was to investigate the persistence of SARS-CoV-2 in diverse tissues at three timepoints following recovery from mild COVID-19, as well as its association with long COVID symptoms. <h3>Methods</h3> This single-centre, cross-sectional cohort study was done at China–Japan Friendship Hospital in Beijing, China, following the omicron wave of COVID-19 in December, 2022. Individuals with mild COVID-19 confirmed by PCR or a lateral flow test scheduled to undergo gastroscopy, surgery, or chemotherapy, or scheduled for treatment in hospital for other reasons, at 1 month, 2 months, or 4 months after infection were enrolled in this study. Residual surgical samples, gastroscopy samples, and blood samples were collected approximately 1 month (18–33 days), 2 months (55–84 days), or 4 months (115–134 days) after infection. SARS-CoV-2 was detected by digital droplet PCR and further confirmed through RNA in-situ hybridisation, immunofluorescence, and immunohistochemistry. Telephone follow-up was done at 4 months post-infection to assess the association between the persistence of SARS-CoV-2 RNA and long COVID symptoms. <h3>Findings</h3> Between Jan 3 and April 28, 2023, 317 tissue samples were collected from 225 patients, including 201 residual surgical specimens, 59 gastroscopy samples, and 57 blood component samples. Viral RNA was detected in 16 (30%) of 53 solid tissue samples collected at 1 month, 38 (27%) of 141 collected at 2 months, and seven (11%) of 66 collected at 4 months. Viral RNA was distributed across ten different types of solid tissues, including liver, kidney, stomach, intestine, brain, blood vessel, lung, breast, skin, and thyroid. Additionally, subgenomic RNA was detected in 26 (43%) of 61 solid tissue samples tested for subgenomic RNA that also tested positive for viral RNA. At 2 months after infection, viral RNA was detected in the plasma of three (33%), granulocytes of one (11%), and peripheral blood mononuclear cells of two (22%) of nine patients who were immunocompromised, but in none of these blood compartments in ten patients who were immunocompetent. Among 213 patients who completed the telephone questionnaire, 72 (34%) reported at least one long COVID symptom, with fatigue (21%, 44 of 213) being the most frequent symptom. Detection of viral RNA in recovered patients was significantly associated with the development of long COVID symptoms (odds ratio 5·17, 95% CI 2·64–10·13, p<0·0001). Patients with higher virus copy numbers had a higher likelihood of developing long COVID symptoms. <h3>Interpretation</h3> Our findings suggest that residual SARS-CoV-2 can persist in patients who have recovered from mild COVID-19 and that there is a significant association between viral persistence and long COVID symptoms. Further research is needed to verify a mechanistic link and identify potential targets to improve long COVID symptoms. <h3>Funding</h3> National Natural Science Foundation of China, National Key R&D Program of China, Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, and New Cornerstone Science Foundation. <h3>Translation</h3> For the Chinese translation of the abstract see Supplementary Materials section.

<h2>Abstract</h2><h3>Background</h3> Various techniques have been reported for the treatment of chronic acromioclavicular (AC) joint separation. The purpose of this study was to evaluate the results of surgical construction of coracoclavicular ligament using coracoid process transfer augmented with a hook plate fixation. <h3>Materials and methods</h3> Twenty-one patients treated with coracoid process transfer augmented with a hook plate fixation for chronic type III and V AC injuries were retrospectively analysed in 2003–2009. The age of the patients ranged from 23 to 58 years with an average age of 41.6 years. The patients were followed up clinically and radiographically, with an average of 33.0 months. Functional status and the ability to return to work were recorded during follow-up. Constant score and visual analogue scale (VAS) for pain were measured. <h3>Results</h3> The mean Constant score has increased from 70.9 points preoperatively to 90.7 points at follow-up. The mean VAS score has decreased from 4.7 preoperatively to 1.2 at follow-up. The average abduction was 172°, forward flexion was 170° and external rotation was 56°. There were 10 excellent results, 10 good results and one fair result. All patients had resumed their job or returned to original sport activity at mean 3.7 months postoperatively. No reduction loss was observed after plate removal and the final follow-up. <h3>Conclusions</h3> Despite retrospective nature of the study, the outcomes of surgical construction with process transfer augmented with hook plate fixation are promising for chronic type III and V AC injuries. <h3>Level of evidence</h3> Therapeutic level IV. Retrospective case series, treatment study.

The purpose of this study was to compare the clinical results of percutaneous reduction and Steinman pin fixation for Sanders II calcaneal fractures with those of operative management through an extensile lateral approach.Fifty-three patients treated with standard open reduction and internal fixation (ORIF group) and 54 patients who had undergone percutaneous reduction and Steinman pin fixation (CRIF group) were retrospectively reviewed. There were no differences between the groups regarding sex, age or fracture classification. Pain and functional outcome were evaluated with a visual analogue scale (VAS) and American Orthopaedic Foot and Ankle Society (AOFAS) scores. Wound complications and radiological results were compared.At a mean follow-up of 40.4 months (24 to 56 months), there were no differences between the two groups in mean AOFAS score, VAS score or radiologically determined variables. Two cases of deep infection and six of poor wound healing occurred in the ORIF group and none in the CRIF group. Subtalar and ankle motion was found to be better in the CRIF group.Percutaneous reduction and Steinman pin fixation minimizes complications and achieves functional outcomes comparable to those of the open techniques in patients with Sanders II calcaneal fractures.

Detailed characteristics of rheumatic symptoms of coronavirus disease 2019 (COVID-19) were still unknown. We aim to investigate the proportions, characteristics, and risk factors of this condition.In this prospective, longitudinal cohort study, discharged patients with COVID-19 were interviewed face-to-face at 12 months after symptom onset. Rheumatic symptoms following COVID-19 included newly occurring joint pain and/or joint swelling. The risk factors of developing rheumatic symptoms were identified by multivariable logistic regression analysis.In total, 1296 of 2469 discharged patients with COVID-19 were enrolled in this study. Among them, 160 (12.3% [95% confidence interval {CI}, 10.6%-14.3%]) suffered from rheumatic symptoms following COVID-19 at 12-month follow-up. The most frequently involved joints were the knee joints (38%), followed by hand (25%) and shoulder (19%). Rheumatic symptoms were independent of the severity of illness and corticosteroid treatment during the acute phase, while elderly age (odds ratio [OR], 1.22 [95% CI, 1.06-1.40]) and female sex (OR, 1.58 [95% CI, 1.12-2.23]) were identified as the risk factors for this condition.Our investigation showed a considerable proportion of rheumatic symptoms following COVID-19 in discharged patients, which highlights the need for continuing attention. Notably, rheumatic symptoms following COVID-19 were independent of the severity of illness and corticosteroid treatment during the acute phase.

Abstract Differently substituted 4‐(1,3‐dithiolan‐2‐ylidene)but‐1‐ynes were conveniently converted into the corresponding thiophenes and dihydrothiophenes in good to high yields under mild conditions. 1,3‐Dithiolan‐2‐ylidene acted as a masked thiolate anion and underwent tandem fragmentation and 5‐ exo ‐ dig annulation with an alkyne moiety to form the five‐membered sulfur heterocycles.

A new and efficient synthetic approach to [<i>gem</i>-bis(alkylthio)vinyl]allenes has been developed involving iron(III)-catalyzed dehydration C(sp²)–C(sp²) coupling of tertiary propargyl alcohols and α-oxo ketene dithioacetals, affording a variety of [<i>gem</i>-bis(alkylthio)vinyl]allenes in good to excellent yields.

Rapid pathogen detection is critical for prompt treatment, interrupting transmission routes, and decreasing morbidity and mortality. The V-type CRISPR system had been used for rapid pathogen detection. However, whether single-stranded DNA in CRISPR system can cause false positives remains undetermined. Herein, we show that high molar concentration of Cas12a effector tolerated more mismatches on ssDNA and activated its trans-cleavage activity at six base matches. Reducing Cas12a and crRNA molar concentration increased the minimal base-match number required for Cas12a ssDNA activation to 11, which reducing nonspecific activation. We then established a Cas12a-based M tuberculosis detection system with a primer having an 8 bp overlap with crRNA. This system did not exhibit primer-induced false positives, and minimum detection copy reached 1 copy/uL (inputting 1-μL sample) in standard strains. The Cas12a-based M tuberculosis detection system showed 80.0% sensitivity and 100.0% specificity in verification using clinical specimens, compared with Xpert MTB/RIF, which showed 72.0% sensitivity and 90.9% specificity. All these results prove that appropriate concentration of cas12a effector can effectively perform nucleic acid detection.

As a severe complication of influenza infection, acute respiratory distress syndrome (ARDS) has higher morbidity and mortality. Although IL-36γ has been proven to promote inflammation at epithelial sites and protect against specific pathogen infection, the detailed roles in severe influenza infection remain poorly understood. In this study, we have found that the expression of IL-36γ is higher in influenza-induced ARDS patients than healthy individuals. IL-36γ was induced in human lung epithelial cells and peripheral blood mononuclear cells by Influenza A virus (IAV) infection and its induction was synergistically correlated with initiation of cyclooxygenase-2 (COX-2)/ Prostaglandin E2 (PGE2) axis. We also have found that expression of superficial IL-36R was elevated in severe influenza patients and in IAV-stimulated cells. Furthermore, although IL-36γ enhanced the induction of type Ⅰ and Ⅲ interferons (IFNs), which promoted IAV-mediated IFN-stimulated STAT1 and STAT2 phosphorylated inhibition in lung epithelial cells, the downstream interferon-stimulated genes (ISGs) were not affected. Finally, we have revealed that IL-36γ treatment could promote apoptosis and inhibit autophagy in the early stages of IAV infection. Overall, these findings demonstrated IL-36γ is a critical host immune factor in response to IAV infection. It has potential activity in the regulation of interferon signaling pathway and involved in different types of programmed cell death in human airway epithelial cells as well.

To identify the association between the kinetics of viral load and clinical outcome in severe coronavirus disease 2019 (COVID-19) patients, a retrospective study was performed by involved 188 hospitalized severe COVID-19 patients in the LOTUS China trial. Among the collected 578 paired throat swab (TS) and anal swab (AS) samples, viral RNA was detected in 193 (33.4%) TS and 121 (20.9%) AS. A higher viral RNA load was found in TS than that of AS, with means of 1.0 × 106 and 2.3 × 105 copies/ml, respectively. In non-survivors, the viral RNA in AS was detected earlier than that in survivors (median of 14 days vs 19 days, P = 0.007). The positivity and viral load in AS were higher in non-survivors than that of survivors at week 2 post symptom onset (P = 0.006). A high initial viral load in AS was associated with death (OR 1.368, 95% CI 1.076–1.741, P = 0.011), admission to the intensive care unit (OR 1.237, 95% CI 1.001–1.528, P = 0.049) and need for invasive mechanical ventilation (OR 1.340, 95% CI 1.076–1.669, P = 0.009). Our findings indicated viral replication in extrapulmonary sites should be monitored intensively during antiviral therapy.

The catalytic intramolecular aromatic C–H alkenylation of arenes with non-activated ketone carbonyls has been realized for the first time, leading to the synthesis of antiviral 4-alkylene quinolin-2-ones, where the quaternary carbon adjacent to the ketone carbonyl plays an essential role in this catalytic reaction.

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

A hierarchical medi-MOF-1 was designed and synthesized, and the pores could accommodate the protein Cyt c with catalytic activity.

Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-Å resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature. Glycosylasparaginase (GA) is an amidase and belongs to a novel family of N-terminal nucleophile hydrolases that use a similar autoproteolytic processing mechanism to generate a mature/active enzyme from a single chain protein precursor. From bacteria to eukaryotes, GAs are conserved in primary sequences, tertiary structures, and activation of amidase activity by intramolecular autoproteolysis. An evolutionarily conserved His-Asp-Thr sequence is cleaved to generate a newly exposed N-terminal threonine, which plays a central role in both autoproteolysis and in its amidase activity. We have recently determined the crystal structure of the bacterial GA precursor at 1.9-Å resolution, which reveals a highly distorted and energetically unfavorable conformation at the scissile peptide bond. A mechanism of autoproteolysis via an N-O acyl shift was proposed to relieve these conformational strains. However, it is not understood how the polypeptide chain distortion was generated and preserved during the folding of GA to trigger autoproteolysis. An obstacle to our understanding of GA autoproteolysis is the uncertainty concerning its quaternary structure in solution. Here we have revisited this question and show that GA forms dimers in solution. Mutants with alterations at the dimer interface cannot form dimers and are impaired in the autoproteolytic activation. This suggests that dimerization of GA plays an essential role in autoproteolysis to activate the amidase activity. Comparison of the melting temperatures of GA dimers before and after autoproteolysis suggests two states of dimerization in the process of enzyme maturation. A two-step dimerization mechanism to trigger autoproteolysis is proposed to accommodate the data presented here as well as those in the literature. glycosylasparaginase aspartylglycosaminuria N-terminal nucleophile autoproteolyzed/mature form of the T152C mutant protein maltose-binding protein dimethyl suberimidate protein intervening sequence Glycosylasparaginase (GA)1 is an amidase involved in Asn-linked glycoprotein degradation (1Aronson N.N., Jr. Kuranda M.J. FASEB J. 1989; 3: 2615-2622Google Scholar). A deficiency in the human GA leads to severe clinical symptoms, known as aspartylglycosaminuria (2Mononen I. Fisher K.J. Kaartinen V. Aronson N.N., Jr. FASEB J. 1993; 7: 1247-1256Google Scholar). GA is widely distributed in vertebrate tissues (3Tollersrud O.K. Aronson N.N., Jr. Biochem. J. 1992; 282: 891-897Google Scholar), insect cells (4Liu Y. Dunn G.S. Aronson N.N., Jr. Glycobiology. 1996; 6: 527-536Google Scholar), and in bacteria (5Tarentino A.L. Plummer T.H., Jr. Biochem. Biophys. Res. Commun. 1993; 197: 179-186Google Scholar). GA joins the proteasome and penicillin acylase as a novel class of enzymes, called N-terminal nucleophile (Ntn) hydrolases that catalytically use a processed N-terminal threonine, serine, or cysteine as both a polarizing base and a nucleophile (6Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C.E. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Google Scholar). The crystal structures of human and bacterial GA show a similar structural frame of an αββα-sandwich, common to all the Ntn hydrolases (7Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nat. Struct. Biol. 1995; 2: 1102-1108Google Scholar, 8Guo H.-C., Xu, Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Google Scholar, 9Xuan J. Tarentino A.L. Grimwood B.G. Plummer T.H., Jr. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Google Scholar, 10Oinonen C. Rouvinen J. Protein Sci. 2000; 9: 2329-2337Google Scholar). One intriguing aspect of GA is that a single chain precursor is processed by intramolecular autoproteolysis that generates the 17-kDa α- and 15-kDa β-subunits and exposes the active site threonine at the newly generated N-terminal end of the β-subunit (11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar, 12Tikkanen R. Riikonen A. Oinonen C. Rouvinen R. Peltonen L. EMBO J. 1996; 15: 2954-2960Google Scholar). A similar mechanism is utilized in protein splicing that involves two concerted autocleavages and one ligation of the polypeptide chain (13Paulus H. Annu. Rev. Biochem. 2000; 69: 447-496Google Scholar, 14Perler F.B. Adam E. Curr. Opin. Biotechnol. 2000; 11: 377-383Google Scholar). Crystal structures of GA precursor have provided us a structural basis to propose a detailed mechanism of intramolecular autoproteolysis (15Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Google Scholar). The GA precursor undergoes a two-step mechanism to break up the polypeptide chain, through an N-O acyl shift and an ester intermediate. In the first step, the nucleophile Thr-152 is activated by a base Asp-151 that is held in an unusual but precise geometry by Thr-203 to deprotonate the nucleophile Thr-152. After the nucleophilic attack, a transitional tetrahedral anion intermediate is stabilized by a polar group of Thr-170 that could further be polarized to carry positive charge by Arg-180, possibly through a bound solvent molecule. Collapse of the tetrahedral anion intermediate shifts the linkage from an amide bond to an ester bond (N-O acyl shift). Usually the equilibrium of N-O acyl shift favors the amide bond, resulting in a peptide bond that does not break often at threonine, serine, or cysteine. However, the strained, tight turn conformation at the scissile peptide bond observed in the GA precursor structures could drive the equilibrium toward an ester bond formation (15Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Google Scholar). The second step of autoproteolysis is a simple hydrolysis of the ester intermediate by a neighboring water molecule, resulting in an active amidase with α- and β-subunits. Recently, both scissile peptide bonds involved in protein splicing of PI-SceI have also been found to be in distortedtrans conformations (16Poland B.W., Xu, M.Q. Quiocho F.A. J. Biol. Chem. 2000; 275: 16408-16413Google Scholar). Similar to GA autoproteolysis, relieving the distorted/strained main chain atoms is also believed to help drive an N-S acyl shift in PI-SceI precursor. It is not understood how the polypeptide chain distortion to trigger autoproteolysis is generated and why these local constraints are not removed during the folding of the GA precursor. It seems plausible that the structural constraints are generated only at a late stage of protein folding, for example, during the formation of the native quaternary structure. Dimerization of GA precursors has been suggested to be a prerequisite to trigger autoproteolysis (8Guo H.-C., Xu, Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Google Scholar, 17Riikonen A. Rouvinen J. Tikkanen R. Julkunen I. Peltonen L. Jalanko A. J. Biol. Chem. 1996; 271: 21340-21344Google Scholar, 18Saarela J. Laine M. Tikkanen R. Oinonen C. Jalanko A. Rouvinen J. Peltonen L. J. Biol. Chem. 1998; 273: 25320-25328Google Scholar). However, the native quaternary structure of GA was controversial. Among several crystal forms, the same dimer structure is observed for the human and bacterial GA, either as precursors or autoproteolyzed enzymes (7Oinonen C. Tikkanen R. Rouvinen J. Peltonen L. Nat. Struct. Biol. 1995; 2: 1102-1108Google Scholar, 8Guo H.-C., Xu, Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Google Scholar, 9Xuan J. Tarentino A.L. Grimwood B.G. Plummer T.H., Jr. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Google Scholar,15Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Google Scholar). However, in solution, GA was reported as monomeric for mammalian and bacterial enzymes (3Tollersrud O.K. Aronson N.N., Jr. Biochem. J. 1992; 282: 891-897Google Scholar, 9Xuan J. Tarentino A.L. Grimwood B.G. Plummer T.H., Jr. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Google Scholar, 11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar, 19Tollersrud O.K. Heiskanen T. Peltonen L. Biochem. J. 1994; 300: 541-544Google Scholar), whereas others reported dimeric GA for the human (17Riikonen A. Rouvinen J. Tikkanen R. Julkunen I. Peltonen L. Jalanko A. J. Biol. Chem. 1996; 271: 21340-21344Google Scholar, 20Kaartinen V. Williams J.C. Tomich J. Yates III, J.R. Hood L.E. Mononen I. J. Biol. Chem. 1991; 266: 5860-5869Google Scholar), chicken (3Tollersrud O.K. Aronson N.N., Jr. Biochem. J. 1992; 282: 891-897Google Scholar), and insect enzymes (4Liu Y. Dunn G.S. Aronson N.N., Jr. Glycobiology. 1996; 6: 527-536Google Scholar). This study was designed to re-examine the native quaternary structure of GA in solution and to study the significance of the quaternary structure in the intramolecular autoproteolysis that is essential to activate the amidase activity for glycoprotein degradation. By using biochemical and biophysical methods, we confirm that GA exists as dimers in solution. Structure-based mutants designed to disrupt the dimer interface lose their ability to form dimers and also lose their ability for autoproteolytic activation of amidase activity. Proteins were expressed and purified by existing procedures (21Cui T. Liao P.-H. Guan C. Guo H.-C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1961-1964Google Scholar), with some modifications. The expression of maltose-binding protein (MBP)-GA fusion proteins was induced in the TB1 cells by adding 1 mmisopropyl-1-thio-β-d-galactopyranoside overnight at 30 °C. Cells were pelleted by centrifugation, resuspended in 20 mm Tris buffer, pH 7.6, 50 mm NaCl, 1 mm EDTA, and lysed by sonication. Fusion proteins were affinity-purified from the crude extracts over an amylose column according to the protocol of the manufacturer (New England Biolabs). All purification procedures were carried out at 4 °C. To obtain GA proteins, the purified fusion proteins were further digested with factor Xa at room temperature overnight, and GA was then separated from MBP and factor Xa with a HiTrap Q-Sepharose (Amersham Biosciences) column. Similar protocols were used to purify autoproteolysis-inactive mutant precursors (T152A). For the precursor proteins of autoproteolysis-active mutant precursors (T152C mutant), 10 mm glycine was included during the entire course of protein expression and purification to inhibit autoproteolysis. Protein concentration was determined by absorbance measurements at 280 nm in 6m guanidine hydrochloride (22Edelhoch H. Biochemistry. 1967; 6: 1948-1954Google Scholar). A 200-μl volume, 20 μm each of the GA or MBP fusion proteins, was applied to an AmershamBiosciences HiPrep 16/60 Sephacryl S-200 HR gel filtration column, equilibrated in a solution containing 10 mmpotassium-phosphate buffer, pH 7.4, and 1 mm EDTA. According to the manufacturer, this column effectively separates globular proteins in the molecular mass range of 5–250 kDa. The gel filtration standards, a mixture of five molecular weight markers, were purchased from Bio-Rad: bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B-12 (1,350 Da). Thus, bovine thyroglobulin was eluted at the point representing the void volume of the column. Cross-linking was performed at 0.1–0.2 mg/ml (1.3–6.2 μm) protein and 0.0005–1% glutaraldehyde in 10 mm potassium-phosphate buffer, pH 7.4, and incubation for 6 h at room temperature. Reactions were quenched with 0.15 volumes of 0.5 m glycine. Cross-linked protein samples were denatured in 1% SDS, 1% β-mercaptoethanol at 90 °C for 10 min, and then analyzed by SDS-PAGE. The gels were stained with Coomassie Blue. Similar experiments were also performed using 1–4 mg/ml dimethyl suberimidate (DMS) as a cross-linking reagent. Sedimentation was performed using a modified version of the method described by Martin and Ames (23Martin R.G. Ames B.N. J. Biol. Chem. 1961; 236: 1372-1379Google Scholar). Linear gradients of 10–30% sucrose in 10 mm potassium-phosphate buffer, pH 7.4, were equilibrated at 4 °C for 2–4 h prior to use. Protein was diluted in the same buffer to 30 μm, preincubated for 30 min at 4 °C, and then layered on the gradients using a 20-gauge needle. Gradients were centrifuged 40,000 rpm for 12 h in a Beckman SW55 Ti rotor. Gradients were withdrawn from the top into a series of 100-μl fractions. Protein peaks in the gradient were determined by Bradford assays of each fraction with reading of absorbance at 595 nm. DNA manipulation and site-directed mutagenesis (Kunkel methods) were carried out as described previously (11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar). Wild-type Flavobacteriumglycosylasparaginase coding sequence was cloned into either the pMAL system or the Litmus system for site-directed mutagenesis, following the manufacturer's protocols (New England Biolabs). DNA clones with desired mutations were confirmed by sequence analyses using an ABI automatic DNA sequencer. CD spectra were recorded from 185 to 250 nm using an AVIV 62DS spectrometer equipped with a thermoelectric sample temperature controller, with a 1-mm path length cuvette. Protein concentrations were 2–5 μm in 10 mm potassium-phosphate buffer, pH 7.4, and 1 mmEDTA and were determined by absorbance measurements in 6 mguanidine hydrochloride (22Edelhoch H. Biochemistry. 1967; 6: 1948-1954Google Scholar). The spectra were collected at 25 °C as the average of 10 scans, using a 15-s integration time at 1.0 nm wavelength increments. Following the buffer base-line correction, the spectra were normalized to the protein concentration and are expressed as molar ellipticity. Thermal unfolding curves were recorded at 222 nm upon heating from 5 to 95 °C at a rate of 60 °C/h, with a 1 °C increment, 30-s accumulation time. The melting temperature (T m) was determined from the differential melting curves d[Θ220]/dT (24John D.M. Weeks K.M. Protein Sci. 2000; 9: 1416-1419Google Scholar). ORIGIN software (Microcal, Inc.) was used for the CD analyses and display. Previous reports concerning the native GA quaternary structure drew conclusions mainly based on the apparent molecular masses on sizing chromatography or native gel electrophoresis, often using samples from cell extracts and visualizing a trace amount of GA by Western blot methods. Contradicting observations were reported from different groups; some concluded GA monomers (3Tollersrud O.K. Aronson N.N., Jr. Biochem. J. 1992; 282: 891-897Google Scholar, 9Xuan J. Tarentino A.L. Grimwood B.G. Plummer T.H., Jr. Cui T. Guan C. Van Roey P. Protein Sci. 1998; 7: 774-781Google Scholar, 11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar, 19Tollersrud O.K. Heiskanen T. Peltonen L. Biochem. J. 1994; 300: 541-544Google Scholar), whereas others reported dimers for GA precursor or autoproteolyzed GA (3Tollersrud O.K. Aronson N.N., Jr. Biochem. J. 1992; 282: 891-897Google Scholar, 4Liu Y. Dunn G.S. Aronson N.N., Jr. Glycobiology. 1996; 6: 527-536Google Scholar, 17Riikonen A. Rouvinen J. Tikkanen R. Julkunen I. Peltonen L. Jalanko A. J. Biol. Chem. 1996; 271: 21340-21344Google Scholar, 20Kaartinen V. Williams J.C. Tomich J. Yates III, J.R. Hood L.E. Mononen I. J. Biol. Chem. 1991; 266: 5860-5869Google Scholar). These contradictory observations have prompted us to re-investigate the quaternary structure of GA more closely using purified precursors and the autoproteolyzed enzyme ofFlavobacterium GA. Wild-type GA spontaneously autoproteolyzes in cells into the mature form amidase with two (α and β) subunits, and thus it is impossible to isolate the precursor of wild-type GA. To study the quaternary structure of purified GA precursor, we thus have to resort to GA mutants. T152A precursor has the same three-dimensional structure as other precursor proteins that are active in autoproteolysis (15Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Google Scholar). Due to the loss of its critical nucleophile for autoproteolysis, we are able to purify a large quantity of precursor protein of the T152A mutant. Autoproteolysis of a second mutant, T152C, can be switched on and off by a reversible inhibitor, glycine (15Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Google Scholar). With the exception of a slower reaction rate, T152C resembles the wild-type GA in autoproteolysis activity (11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar, 25Guan C. Liu Y. Shao Y. Cui T. Liao W. Ewel A. Whitaker R. Paulus H. J. Biol. Chem. 1998; 273: 9695-9702Google Scholar), and the autoproteolyzed T152C enzyme (T152Cm) has an identical crystal structure to that of the wild-type mature enzyme (8Guo H.-C., Xu, Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Google Scholar). Throughout this study, the T152A precursor was used to represent a native precursor structure, whereas the wild-type and/or mature T152Cm proteins were used to represent a native structure of mature GA amidase after autoproteolysis. The purified GA proteins were first examined by chromatography on a gel filtration column. Because the native autoproteolyzed amidase still has the two newly generated (α and β) subunits in tight association (26Riikonen A. Tikkanen R. Jalanko A. Peltonen L. J. Biol. Chem. 1995; 270: 4903-4907Google Scholar, 27Tarentino A.L. Quinones G. Hauer C.R. Changchien L.M. Plummer T.H., Jr. Arch. Biochem. Biophys. 1995; 316: 399-406Google Scholar), a single chain GA precursor and a native autoproteolyzed amidase should have similar calculated molecular masses of 32 kDa. As shown in Fig. 1 A, autoproteolyzed amidase of wild-type GA or T152Cm protein was eluted as a single peak with an apparent molecular mass of 43 kDa, when compared with a mixture of gel filtration size standards (Bio-Rad). Thus, the apparent molecular mass of mature GA was about 40% larger than a GA monomer of 32 kDa. Similarly, purified precursor T152A protein was eluted as a single peak with an apparent molecular mass of about 1.4-fold of a monomer. The precursor was eluted at a slightly larger volume than the mature enzyme. This is consistent with an uncleaved loop in the precursor that becomes cleaved and thus more floppy and bulky in the mature enzyme. Interestingly, a 43-kDa recombinant maltose-binding protein (MBP2 from New England Biolabs) was eluted with a substantially smaller apparent molecular mass (about 25 kDa) when compared with the same size standards (Fig. 1 B). All these observations demonstrated the limitations of using gel filtration chromatography to estimate molecular masses of macromolecules, because the mobility of proteins in gel filtration columns was affected not only by molecular weight and size but also by molecular shape. There are various ways to interpret the single peaks of GA on chromatograms with elution volumes at the middle between those expected for monomers and dimers. These single peaks could represent GA monomers with a smaller retention volume when compared with the size standards, which may be due to an extensive hydrophobic surface observed in the crystal structures. These single peaks could also be interpreted as GA dimers with a larger retention volume than expected, which may be due to the compact shape of the dimer observed in the crystal structures (8Guo H.-C., Xu, Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Google Scholar). A third explanation for the unusual mobility is that GA dimers are in fast equilibrium with monomers during the run through the gel filtration column. This uncertainty appears to be the source of confusion that results in contradictory reports in the literature. In an attempt to demonstrate the sensitivity of gel filtration chromatography to the shape of proteins, we re-examined the behavior of GA fusion proteins on the gel filtration column. We reasoned that attaching an MBP to the N-terminal end of GA protein would change the shape of a monomer from a quite compact GA structure to an irregular shape of a fusion protein. Therefore, if existing as a single species in solution, these fusion proteins might behave differently from GA protein alone and thus provide better resolution to distinguish monomers from dimers. Otherwise, if GA dimers are in equilibrium with monomers, the fusion proteins would still be eluted as single peaks with apparent molecular masses of about 1.4-fold of a monomer. Fig. 1 B shows the gel filtration chromatograms for purified MBP-GA fusion proteins, before and after the GA domain was autoproteolyzed. As a control, MBP protein alone was eluted as a single peak with an apparent molecular mass of 25 kDa, which is smaller than that calculated for a monomer of 43 kDa. Nonetheless, it indicates that MBP does not dimerize in solution. For a monomeric MBP-GA fusion protein, either as a precursor or an autoproteolyzed amidase, the calculated molecular mass is 75 kDa. The MBP-T152A (GA precursor) fusion protein was eluted as a single peak, with an apparent molecular mass of 138 kDa. Likewise, MBP fusion with T152C mature GA (MBP-T152Cm) was eluted with an apparent molecular mass of 138 kDa that is 1.8-fold of the calculated molecular mass for a monomer. This shift of molecular mass from 1.4- to 1.8-fold of a monomer indicates that the unusual mobility of GA is not a result of fast equilibrium. Thus, considering that MBP alone was eluted at 0.6-fold of its calculated molecular weight, the MBP-GA fusion proteins eluted at 1.8-fold of the calculated molecular masses are more likely to be dimers than monomers. These results also suggest that the dimerization of fusion proteins is mediated by the GA domain and not the MBP domain. To confirm GA dimers in solution, we performed cross-linking with glutaraldehyde and DMS. Cross-linking of T152A precursor with 0.001–1% glutaraldehyde (amine-amine cross-linking) at room temperature and 0.1 mg/ml (3.1 μm) protein indicated that the GA precursor was dimeric under these conditions (Fig. 2). Following SDS-PAGE, the T152A precursor yielded a characteristic pattern of two bands corresponding to cross-linked monomer and dimer (Fig. 2 A), whereas an uncross-linked control (i.e. without glutaraldehyde treatment) gave a single band corresponding to a monomer. Similar cross-linking results were obtained with autoproteolyzed enzyme of wild-type GA (Fig. 2 B), although the pattern is more complicated because there are two subunits, α and β, in each native mature/autoproteolyzed enzyme. Without cross-linking treatment, the wild-type mature enzyme gave two bands for the α- and β-subunits. A low dose of cross-linking reagent (0.001–0.01%) gave a new cluster of bands of 30–34 kDa corresponding to two cross-linked subunits either from the same GA molecule (α-β and α′-β′ of 32 kDa) or through the dimer interface (α-α′, α-β′, α′-β, and β-β′ ranging from 30 to 34 kDa). At higher concentrations of cross-linking treatment (0.1–1%), an additional cluster of bands was present corresponding to completely cross-linked dimer of autoproteolyzed enzyme (∼64 kDa of α-β-α′-β′). No band corresponding to three cross-linked subunits (e.g. α-β-α′, etc.) appeared. This might be due to a preferential cross-linking of contacts either through dimer interface or within a monomer. Thus, by the time two subunits were cross-linked through the minor cross-linking surface, they were also cross-linked through the major cross-linking surface, resulting in the 64-kDa species. The cross-linking pattern of mature T152Cm protein (Fig. 2 C) is the same as the wild-type enzyme and confirms a dimeric structure of mature enzyme. Similar results were also obtained using DMS as the cross-linking reagent (data not shown). All together, results here confirm that both GA precursor and mature enzyme form dimers in solution. To examine the significance of dimerization in autoproteolysis, we designed and generated mutants that would disrupt dimer interactions without affecting protein folding. With the aid of the crystal structures of the GA precursor protein (15Xu Q. Buckley D. Guan C. Guo H.-C. Cell. 1999; 98: 651-661Google Scholar) and autoproteolyzed amidase (8Guo H.-C., Xu, Q. Buckley D. Guan C. J. Biol. Chem. 1998; 273: 20205-20212Google Scholar), we identified several targets at the dimerization interface for mutagenesis, hoping to weaken or disrupt the dimer formation (Fig. 3 A). In the dimers, Val-105 is sequestered out of the solvent to form hydrophobic contacts with its equivalent from the other monomer. Similarly, Ile-186 from each monomer interacts with each other at the dimer interface. In addition, conserved residue Glu-220 forms charge interactions with conserved residue Arg-238 of the adjacent monomer. All of these residues point away from the structural core of the monomer and thus are unlikely to disturb protein packing of each monomer. Furthermore, because these four residues are located on the opposite side of the scissile peptide bond (Fig. 3 A), they are not likely to be involved directly in the autoproteolysis. Therefore, replacement of each of these residues was hypothesized to destabilize dimer formation but not to affect the folding of the monomer or to change active site residues. Four dimer interface mutants were generated to study the effect of mutation at the dimer interface, V105K, I186D, E220K, and R238D. These mutant proteins were also expressed as MBP fusion proteins by similar protocol published previously (11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar) for the wild-type and other GA mutants. Purification of GA interface mutants alone proved to be difficult because they were unstable and very sensitive to protease degradation during the process of protein purification, particularly after factor Xa digestion to separate GA mutants from the MBP domain. However, when purified as MBP fusion proteins, these mutants were more stable and thus allowed us to obtain sufficient amounts of proteins for further characterization. Nonetheless, there are still a few minor degradation bands that were eluted together with the MBP-GA full-length protein from a maltose-affinity column (Fig. 3 B, lanes 11–18). This indicates these minor degradation products may still retain an intact MBP domain but with a degraded GA mutant domain. This is in contrast to the wild-type GA or other active site mutants that are very stable by the same expression and purification procedure. As shown in Fig. 3 B, wild-type GA spontaneously autoproteolyzes into α- and β-subunits during protein purification (lane 2). The T152C mutant also partially autoproteolyzes (lane 3) during the protein purification, resulting in the same α- and β-subunits as the wild-type GA. Furthermore, it has been shown that autoproteolysis of T152C mutant can be accelerated by incubation with hydroxylamine (lane 4; see Ref. 11Guan C. Cui T. Rao V. Liao W. Benner J. Lin C.L. Comb D. J. Biol. Chem. 1996; 271: 1732-1737Google Scholar), indicating a thioester intermediate involved in the autoproteolysis of the T152C mutant. Conversely, T152A mutant loses its critical nucleophile for autoproteolysis (lane 5), and hydroxylamine does not have any effect on autoproteolysis of the T152A mutant (lane 6). GA retains the same autoproteolytic activity when purified as MBP fusion proteins. MBP-T152C fusion protein partially autoproteolyzes into MBP-α and -β subunits (Fig. 3 B, lane 7), and hydroxylamine can also stimulate its autoproteolytic activity (lane 8). MBP-T152A fusion does not autoproteolyze either without or with hydroxylamine (lanes 9 and 10). Thus, expression of GA as MBP fusion proteins does not alter their autoproteolytic activity but can stabilize the dimer interface mutants that are extremely sensitive to protease degradation if isolated as individual GA mutants. We therefore characterized all the dimer interface mutants as MBP fusion proteins and compared them to the controls of MBP-T152A precursor and autoproteolyzed MBP-T152C (MBP-T152Cm) as precursor and mature enzyme, respectively.

Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.

Triptolide (TP) exhibits numerous biological activities, including immunosuppressive, anti‑inflammatory and antitumor effects. The aim of the present study was to investigate the role of TP as a potent therapeutic drug for the treatment of lung cancer and to investigate the underlying therapeutic mechanisms. Western blot analyses and reverse transcription‑quantitative polymerase chain reaction (PCR) were performed to investigate the expression of genes at transcriptional and translational levels, respectively. Methylation‑specific PCR assays were conducted to investigate whether TP affects the Wnt inhibitory factor‑1 (WIF‑1) methylation status and subsequently affects apoptosis, migration or the invasion of lung cancer cells. The results of the present study revealed that the methylation status of WIF‑1 in lung cancer cell lines A549 and H460 was significantly enhanced compared with the human normal bronchial epithelial cell line HBE, whereas treatment with TP was revealed to induce the demethylation of WIF‑1. The present study aimed to investigate whether the biological activities of TP are regulated by inhibiting the Wnt signaling pathway via an increase in WIF‑1 expression levels. The results of the present study revealed that Wnt signaling was suppressed in cells following treatment with TP, which was concluded by the downregulation of Axin 2 and β‑catenin expression. Further investigation demonstrated that the silencing of WIF‑1 expression with small interfering RNA reversed the TP‑induced upregulation of WIF‑1 expression, upregulated Axin 2 and β‑catenin expression and enhanced the activation of Wnt signaling. Notably, an upregulation of cellular tumor antigen p53 expression, and downregulation of matrix metalloproteinase‑9 (MMP‑9) and phosphorylated‑nuclear factor‑κB (NF‑κB) P65 (p‑P65) levels was observed following TP treatment. These results suggest that the Wnt, p53 and NF‑κB signaling pathways mediate the potent antitumor effects of TP. Notably, the silencing of WIF‑1 did not completely recover the levels of p53, MMP‑9 and p‑P65 in cells treated with TP compared with the control cells, thus suggesting that TP exhibits further functions in addition to the targeting of WIF‑1.

A series of novel 8-azapurine carbocyclic nucleoside hydrazones were synthesized through a useful procedure starting from amino alcohol and pyrimido dichloride. All the products were characterized by (1)H NMR, (13)C NMR and HRMS spectral analysis and the stereochemical structure of key intermediate was also confirmed by a single crystal X-ray diffraction crystallographic analysis. Moreover, the anticancer activities were evaluated in vitro against human liver cancer Huh-7 cell line and human breast cancer A549 cell line.

Abstract An environmentally friendly route to fully substituted 4,5‐dihydropyrroles was developed through the iron‐catalyzed [4C+1N] cyclization of 4‐acetylenic ketones with primary amines in 1,5‐pentanediol.

Abstract A new and straightforward base‐catalyzed 5‐ exo ‐ trig cyclization of 2‐propynyl‐1,3‐dicarbonyl compounds leads to the corresponding stereodefined 2‐methylene‐2,3‐dihydrofurans in excellent yields.

Abstract OBJECTIVE To identify the anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) in coronavirus disease 2019 (COVID-19) and its relationship with the severity and clinical outcomes of COVID-19. DESIGN Retrospective cohort study. SETTING Three hospitals in China. PARTICIPANTS 274 adult inpatients diagnosed with COVID-19 according to the Protocol for Prevention and Control of COVID-19 (Edition 7) of China and confirmed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) RNA testing, were included from three hospitals from Wuhan, Harbin and Beijing, China from 1 December 2019 to 19 April 2020. The Biobank of Myositis Registry Department of Rheumatology, Peking Union Medical College Hospital, provided the plasma of five patients with anti-MDA5 Ab-related dermatomyositis as positive control group. Demographic, clinical and laboratory data were collected from medical records. The anti-MDA5 Ab was determined by an ELISA assay and was verified by immunoblotting analysis. MAIN OUTCOME MEASURES In hospital death of all cause. RESULTS The positive rate of anti-MDA5 Ab in patients with COVID-19 was 48.2% (132/274) and the anti-MDA5 Ab positive patients tended to represent with severe disease (88.6% vs 66.9%, P &lt;0.0001). The titer of anti-MDA5 Ab was significantly elevated in the non-survivals (5.95±5.16 vs 8.22±6.64, P =0.030) and the positive rate was also higher than that in the survivals (23.5% vs 12.0%, P =0.012). Regarding to severe COVID-19 patients, we found that high titer of anti-MDA5 Ab (≥10.0 U/mL) was more prevalent in the non-survivals (31.2% vs 14.0%, P =0.006). Moreover, early profiling of anti-MDA5 Ab could distinguish severe patients from those with non-severe ones. CONCLUSION Anti-MDA5 Ab was prevalent in the COVID-19 patients and high titer of this antibody is correlated with severe disease and unfavorable outcomes. Early screening and serially monitoring of anti-MDA5 Ab titer have the potential to predict the disease progression of COVID-19.

This study aimed to explore the influences of microRNA-195 (miRNA-195)/Rap2C/MAPK in the proliferation and apoptosis of small cell lung cancer (SCLC) cells. QRT-PCR analysis were executed to evaluate miRNA-195 expression in lung cancer tissues and SCLC cells, and the western blot was implemented to monitor Rap2C protein level and uncovered whether the MAPK signaling pathway in lung cancer tissues and SCLC cells was activated. The CCK-8 experiment was performed to detect cell proliferation ability, and the flow cytometry was utilized to examine cell apoptosis level. Luciferase reporter gene system was executed to disclose the interaction between miRNA-195 and Rap2C. Subcutaneous implantation mouse models of SCLC cells were constructed to detect cell proliferation in vivo, and Kaplan-Meier method calculated patient survival. The expression of Rap2C was higher in lung cancer tissues and SCLC cells than in normal tissues and cells, while the expression of miRNA-195 was lower in lung cancer tissues and SCLC cells than in normal tissues and cells. miRNA-195 lower expression predicted showed reduced overall survival in lung cancer patients. Further loss of function and enhancement experiments revealed that miRNA-195 overexpression could significantly inhibit SCLC cell proliferation and promote cell apoptosis by upregulation of Bax and down-regulation of bcl-2; Luciferase reporter assay demonstrated that miRNA-195 could bind to Rap2C mRNA and inhibit its expression, Rap2C overexpression also related to the poorer prognosis of lung patients. Knockdown of Rap2C suppressed cell proliferation and expedited apoptosis. In addition, overexpression of Rap2C reversed miRNA-195-induced apoptosis and proliferation inhibition. Furthermore, miRNA195 prohibited the activation of MAPK signaling pathway by down-regulating Rap2C. These consequences indicated that miRNA-195 promotes the apoptosis and inhibits the proliferation of small cell lung cancer (SCLC) cells via inhibiting Rap2C protein-dependent MAPK signal transduction

Abstract Background The impact of metagenomic next-generation sequencing (mNGS) on antimicrobial stewardship in patients with lower respiratory tract infections (LRTIs) is still unknown. Methods This retrospective cohort study included patients who had LRTIs diagnosed and underwent bronchoalveolar lavage between September 2019 and December 2020. Patients who underwent both mNGS and conventional microbiologic tests were classified as the mNGS group, while those with conventional tests only were included as a control group. A 1:1 propensity score match for baseline variables was conducted, after which changes in antimicrobial stewardship between the 2 groups were assessed. Results A total of 681 patients who had an initial diagnosis of LRTIs and underwent bronchoalveolar lavage were evaluated; 306 patients were finally included, with 153 in each group. mNGS was associated with lower rates of antibiotic escalation than in the control group (adjusted odds ratio, 0.466 [95% confidence interval, .237–.919]; P = .02), but there was no association with antibiotic de-escalation. Compared with the control group, more patients discontinued the use of antivirals in the mNGS group. Conclusions The use of mNGS was associated with lower rates of antibiotic escalation and may facilitate the cessation of antivirals, but not contribute to antibiotic de-escalation in patients with LRTIs.

The properties of mRNA lipid nanoparticles (mRNA-LNPs), including size, empty particles, morphology, storage stability, and transfection potency, are critically dependent on the preparation methods. Here, a Two-step tangential-flow filtration (TFF) method was successfully employed to improve the properties of mRNA-LNPs during the preparation process. This method involves an additional ethanol removal step prior to the particle fusion process. Notably, this innovative approach has yielded mRNA-LNPs with larger particles, a reduced proportion of empty LNPs, optimized storage stability (at least 6 months at 2–8 °C), improved in vitro transfection efficiency, and minimized distribution in the heart and blood in vivo. In summary, this study represents the implementation of the innovative Two-step TFF method in the preparation of mRNA-LNPs. Our findings indicate substantial enhancements in the properties of our mRNA-LNPs, specifically with regard to the percentage of empty LNPs, stability, transfection efficiency, and in vivo distribution. These improvements have the potential to optimize their industrial applicability and expand their clinical use.

Abstract Active retrotransposons play important roles during evolution and continue to shape our genomes today, especially in genetic polymorphisms underlying a diverse set of diseases. However, studies of human retrotransposon insertion polymorphisms (RIPs) based on whole-genome deep sequencing at the population level have not been sufficiently undertaken, despite the obvious need for a thorough characterization of RIPs in the general population. Herein, we present a novel and efficient computational tool called Specific Insertions Detector (SID) for the detection of non-reference RIPs. We demonstrate that SID is suitable for high-depth whole-genome sequencing data using paired-end reads obtained from simulated and real datasets. We construct a comprehensive RIP database using a large population of 90 Han Chinese individuals with a mean ×68 depth per individual. In total, we identify 9342 recent RIPs, and 8433 of these RIPs are novel compared with dbRIP, including 5826 Alu, 2169 long interspersed nuclear element 1 (L1), 383 SVA, and 55 long terminal repeats. Among the 9342 RIPs, 4828 were located in gene regions and 5 were located in protein-coding regions. We demonstrate that RIPs can, in principle, be an informative resource to perform population evolution and phylogenetic analyses. Taking the demographic effects into account, we identify a weak negative selection on SVA and L1 but an approximately neutral selection for Alu elements based on the frequency spectrum of RIPs. SID is a powerful open-source program for the detection of non-reference RIPs. We built a non-reference RIP dataset that greatly enhanced the diversity of RIPs detected in the general population, and it should be invaluable to researchers interested in many aspects of human evolution, genetics, and disease. As a proof of concept, we demonstrate that the RIPs can be used as biomarkers in a similar way as single nucleotide polymorphisms.

To investigate the mixing amount of graphene oxide and water cement ratio on the microstructure and mechanical properties of graphene oxide reinforced cement based composite material, graphene oxide suspension was developed using improved Hummers method, and the structure, size and morphology of graphene oxide were represented using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and AFM. The results demonstrated that the bending and compressive strength of graphene oxide reinforced cement based composite material improved firstly and then declined with the increase of the mixing amount of graphene oxide, and moreover the improvement of the bending strength was obvious than that of the compressive strength. When the content of graphene oxide was 0.03%, the bending strength reached the maximum, 13.73 MPa. Under a high water cement ratio, the addition of graphene oxide was more effective in enhancing the strength of cement mortar. The representation of the microstructure of cement based composite material with scanning electron microscope (SEM) suggested that graphene oxide could optimize the microstructure of cement hydration products, improve the pore structure of set cement, reduce the volume of micropore in set cement, and increase the compactness of set cement, i.e. apparently strengthen the toughening effect of set cement. The research achievements are useful to improve the mechanical properties of cement based composite materials.

Signature amino acids of H7N9 influenza A virus play critical roles in human adaption and pathogenesis, but their dynamic variation is unknown during disease development. We sequentially collected respiratory samples from H7N9 patients at different timepoints and applied next-generation sequencing (NGS) to the whole genome of the H7N9 virus to investigate the variation at signature sites. A total of 11 patients were involved, from whom 29 samples were successfully sequenced, including samples from multiple timepoints in 9 patients. Neuraminidase (NA) R292K, basic polymerase 2 (PB2) E627K, and D701N were the 3 most dynamic mutations. The oseltamivir resistance-related NA R292K mutation was present in 9 samples from 5 patients, including 1 sample obtained before antiviral therapy. In all patients with the NA 292K mutation, the oseltamivir-sensitive 292R genotype persisted and was not eliminated by antiviral treatment. The PB2 E627K substitution was present in 18 samples from 8 patients, among which 12 samples demonstrated a mixture of E/K and the 627K frequency exhibited dynamic variation. Dual D701N and E627K mutations emerged but failed to achieve predominance in any of the samples. Signature amino acids in PB2 and NA demonstrated high polymorphism and dynamic variation within individual patients during H7N9 virus infection.

Revision internal fixation for femoral neck nonunion in young patients can pose a surgical challenge. Hip salvage protocols include osteotomy and osteosynthesis using various implants and grafting techniques (muscle pedicle, vascularised or nonvascularised fibular graft). The purpose of this study was to evaluate the clinical outcomes of vascularised iliac grafting for femoral neck nonunion in patients younger than 50 years.A total of 22 patients underwent vascularised iliac grafting and internal fixations for femoral neck nonunion were retrospectively analysed. Their mean age was 36.6 years. The cases were evaluated radiographically and clinically.All the patients were followed-up for an average of 64.6 months. The mean time to union for all patients was 5.4 months. The mean Harris Hip Score had increased from 55.3 preoperatively to 85.2 at the latest follow-up. The average neck-shaft angle had changed from 127.4° preoperatively to 128.3° postoperatively. Postoperative progression of osteonecrosis of the femoral head was seen in 3 patients, 2 patients were pain free and 1 patient required total hip arthroplasty 9 years after the revision procedure. There was no further progression in 3 patients with preoperative radiological evidence of the femoral head osteonecrosis.This study indicates that vascularised iliac grafting is a viable option in treatment of femoral neck nonunion in the young adult.

The synthesis of alkyl boronic esters has attracted much attention because of their wide applications in organic synthesis, materials and medicines.Transition-metal catalyzed hydroboration of alkenes was one of the most effective methods to construct alkyl boronic esters.Compared with rhodium, ruthenium, palladium, iridium and other precious metal catalysts, iron, cobalt and nickel catalysts were not only low cost, but also they displayed unique reactivity and selectivity.In this paper, the important advances in hydroboration of alkenes catalyzed by iron, cobalt and nickel have been summarized since 1994, including catalytic activity, selectivity and substrate scope of different catalytic systems.

In order to increase the accuracy and stability of the classical response surface method and relevant method, a new improved response surface method based on the idea of double weighting factors and vector projection method is proposed. The response surface is fitted by the weighted regression technique, which allows the sampling points to be weighted by their distance from the true failure surface and that from the estimated design point. It uses the vector of the gradient projection method to get new sampling points in the process of iteration, in order to make the sampling points closer to the design point, and the value of deviation coefficient is constantly adjusted. To some extent, these strategies increase the accuracy and stability of the response surface method, while the calculation time is decreased. At last, the rationality and efficiency of the proposed method are demonstrated through five examples. Besides, as revealed from this investigation, compared with other conventional algorithms, this method has a few obvious advantages; this algorithm not only has high precision and efficiency, but also has solid stability.

Proximal humerus fractures are commonly observed in postmenopausal women. The goal of this study was to investigate menopause-related changes in cortical structure of the humeral head.Clinical computed tomography (CT) scans of 75 healthy women spanning a wide range of ages (20-72 years) were analyzed. For each subject, cortical bone mapping (CBM) was applied to create a color three-dimensional (3D) thickness map for the proximal humerus. Nine regions of interest (ROIs) were defined in three walls of the humeral head. Cortical parameters, including the cortical thickness (CTh), cortical mass surface density (CM), and the endocortical trabecular density (ECTD), were measured.Compared to premenopausal women, postmenopausal women were characterized by a significantly lower CTh and CM value in the lateral part of the greater tuberosity. Similar changes were only found in ROI 4, but not in ROIs 5-6 in the lesser tuberosity. Linear regression analysis revealed that the CTh and CM value of ROIs 1, 3, and 4 were negatively associated with age. These results showed that menopause-related loss in CTh and CM was mainly in the greater tuberosity besides the proximal part of the lesser tuberosity. Trabecular bone variable measured as ECTD showed a notably lower value in ROIs 1-9 in postmenopausal vs. premenopausal group. Inverse linear associations for ECTD and age were found in ROIs 2, 3, 5, 6, 7, and 9, indicating no site-specific differences of endocortical trabecular bone loss between the greater and lesser tuberosity.Menopause-related cortical loss of the humeral head mainly occurred in the lateral part of the greater tuberosity. The increased rate of humeral bone loss in the greater tuberosity may contribute materially to complex proximal humerus fractures.

Incidence of proximal humeral fractures dramatically increased over the last decade due to demographic changes. The goal of this study was to analyze regional characteristics of cortical bone in the humeral head region using cortical bone mapping (CBM) technique.The proximal humerus of 103 healthy volunteers was imaged by clinical computed tomography (CT) scans. Three groups of volunteers were identified according to age: group A (20-39years), group B (40-59years), and group C (>60years). CBM was applied to create color 3D thickness maps for each proximal humerus. Cortical parameters, including the cortical thickness (CTh), cortical mass surface density (CM), and the endocortical trabecular density (ECTD) were measured over humeral head region after nine regions of interest (ROI) were defined.Cortical bone structure of the humeral head region varied dramatically in cross-section independent of age, with significant cortical thinning at the posterior wall. Particularly, notable cortical thinning was also found in the distal end of lateral wall at 60years of age or older. The degree of regional variability of cortical properties within lateral wall tended to diminish with age.Substantial regional variations in cortical bone structure at humeral head region were observed quantitatively in this study. These morphologic data provided useful information on clinical medicine such as surgical fixation.

As a member of the tripartite motif family, tripartite motif-containing protein 59 (TRIM59) serves as an E3 ubiquitin ligase in various cellular processes, including intracellular signaling, development, apoptosis, protein quality control, innate immunity, autophagy and carcinogenesis. The present study aimed to investigate the expression and prognostic value of TRIM59 in patients with non-small cell lung cancer (NSCLC). Expression of TRIM59 in patients with NSCLC was measured by immunohistochemistry in tissue microarrays. Datasets from The Cancer Genome Atlas (TCGA) were used to further verify the expression level of TRIM59 in NSCLC, lung adenocarcinoma and lung squamous cell carcinoma (LUSC). The prognostic value of TRIM59 in NSCLC was also analyzed. Immunohistochemistry revealed that TRIM59 was primarily located in the cytoplasm of tumor cells. Analysis of TCGA datasets revealed that TRIM59 was more highly expressed in tumor tissues than in normal tissues (P<0.0001). Furthermore, the TRIM59 expression level was associated with tumor differentiation (P=0.012), while no association was observed between TRIM59 expression and any other clinicopathological parameters. However, the average overall survival rate of patients with NSCLC in the high TRIM59 expression group was significantly lower than that in the low expression group (P=0.014), especially in patients with LUSC (P=0.016) and patients with poor differentiation (P=0.033). The multivariate analysis indicated that high TRIM59 expression is an independent prognostic factor in patients with NSCLC (P=0.018) and was associated with poor prognosis in patients with NSCLC. Therefore, TRIM59 may serve as a novel molecular biomarker to predict the prognosis of patients with NSCLC.

N-arylsulfonyl hydrazones were a kind of stable diazo surrogates in organic synthesis.Among them, N-tosylhydrazones occupied main position in the research of carbene chemistry, and many reactions involving N-tosylhydrazones and reviews were published.In recent years, the chemical reactions involving electron-withdrawing groups (EWG)-substituted N-arylsulfonylhydrazones as milder diazo surrogates have been developed rapidly.However, these reactions were not summarized.Therefore, this review focuses on the research progress on EWG-substituted N-arylsulfonylhydrazones used as diazo surrogates in organic synthesis reactions, and emphasized the coupling, cyclization, insertion, multicomponent and Doyle-Kirmse reaction involved N-o-nitrobenzenesulfonylhydrazides and N-o-triftosylhydrazones.